 |  | Simple Western and the Race to Develop a Vaccine for the COVID-19 Coronavirus | [show details] |
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As the world finds itself in the middle of the worst pandemic in a century, finding a vaccine to the deadly COVID-19 coronavirus could not be more urgent. Dr. Ferrari and his colleagues at DIOSynVax (Digitally designed, Immune Optimised Selected and Synthesized Vaccines) are working to
develop a DNA vaccine, and Simple Western from ProteinSimple is an important part of that process. |
 |  | Application Brief 1001: p-EGFR NanoPro Assay | [show details] |
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The epidermal growth factor receptor (EGFR) is a transmembrane receptor for the epidermal growth factor family (EGF-family) of extracellular protein ligands. Ligand binding activates its intracellular tyrosine kinase domain resulting in auto as well as substrate phosphorylation. Mutation and deregulation of EGFR is implicated in many cancer types. The data shows a time dependent change in anti-phospho EGFR (Y1068) antibody signal in HeLa cells in response to EGF treatment. |
 |  | Application Brief 1002: AKT NanoPro Assay | [show details] |
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AKT, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis. This protein kinase is activated by insulin and various growth and survival factors to function in a Wortmannin-sensitive pathway involving PI3 kinase. Activation at Thr309 and Ser473 are the main activating phosphorylation events for AKT. The main isoforms identified so far are AKT1, 2 and 3. AKT3 is mainly expressed in the brain. AKT1 and 2 play differential roles in glucose homeostasis. Our data show increased phosphorylation of AKT using a phospho-Ser743 specific antibody. This antibody is believed to recognize phospho-Ser473 on all three AKT isoforms (see Cell Signaling Technologies data sheet). |
 |  | Application Brief 1003: PAK2 NanoPro Assay | [show details] |
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The p21 activated kinases (PAK) proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1, and have been implicated in a wide range of biological activities. PAK2 is a member of the PAK subfamily 1 including PAK1 (α-PAK), PAK2 (γ-PAK, PAKθ, hPAK65), and PAK3 (β-PAK). The kinase domains within a subfamily show a high degree of sequence identity, and all PAK proteins bind GTP-bound Rho family members at the amino-terminal p21-binding domain (PBD). Our data in HeLa cells show a pattern of 5 peaks using a pan PAK1/2/3 antibody of which two are picked up consistently by 3 different specific PAK2 antibodies identifying them as PAK2 peaks. |
 |  | Application Brief 1004: ALAS1 Loading Control NanoPro Assay | [show details] |
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The human housekeeping protein Delta-aminolevulinate Synthase catalyzes the condensation of glycine with succinyl-CoA to form delta-aminolevulinic acid. It is represented in the Nanopro assay by two peaks at pI's around 5.6 and 5.9. Our data show its utility as a loading control for EGF stimulation in HeLa and MCF10A cells. |
 |  | Application Brief 1005: GSK3α NanoPro Assay | [show details] |
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GSK3 is a critical downstream element of the PI3 kinase/AKT cell survival pathway whose activity can be inhibited by AKT-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β. While GSK-3α and GSK-3β have high sequence homology, their biological function differs. The data presented shows an increase of peaks detected by anti-phospho GSK-3α antibody as well as an increase of the same peaks detected by the total anti-GSK-3α antibody in response to EGF treatment in MCF10A cells. At the same time, the peaks not recognized by the anti-phospho Ser21 antibody decrease in size, implying that these peaks represent non-phospho or non-pS21 phospho GSK-3α forms. The position of these peaks around pI 9 is in accordance with the theoretical pI for this sequence. In other cell systems (A549 for example), both the anti-total GSK-3 antibody as well as the anti-phospho GSK-3α antibody also recognize peaks around 6.0. Alignment of the GSKα and GSKβ profiles in addition to use of antibodies recognizing both isoforms confirmed the specificity of the anti-GSK-3α versus the anti-GSK-3β antibodies used (data not shown). |
 |  | Application Brief 1006: GSK3β NanoPro Assay | [show details] |
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GSK3 is a critical downstream element of the PI3 kinase/AKT cell survival pathway whose activity can be inhibited by AKT-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β. While GSK-3α and GSK-3β have high sequence homology, their biological function differs. The data presented shows an increase of peaks detected by anti-phospho GSK-3β antibody as well as an increase of the same peaks detected by the total anti-GSK-3β antibody in response to EGF treatment in MCF10A cells. At the same time, the peaks not recognized by the anti-phospho Ser21 antibody decrease in size, implying that these peaks represent non-phospho or non-pS21 phospho GSK-3α forms. The position of these peaks around pI 9 is in accordance with the theoretical pI for this sequence. Alignment of the GSKα and GSKβ profiles, in addition to use of antibodies recognizing both isoforms, confirmed the specificity of the anti-GSK-3α versus the anti-GSK-3β antibodies used (data not shown). |
 |  | Application Brief 1007: Thioredoxin1 Loading Control NanoPro Assay | [show details] |
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Thioredoxin acts as an antioxidant and is found in nearly all known organisms. It exists in two isoforms and presents a double peak around pI 4.6. The data presented show the utility of Thioredoxin as a loading control for EGF treated A549 and HeLa cells. |
 |  | Application Brief 1008: Hsp70 NanoPro Assay | [show details] |
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The 70 kilodalton heat shock proteins (Hsp70) are a family of ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms. The Hsp70s are an important part of the cell's machinery, and help to protect proteins from misfolding under stress. We show an increase of Hsp-70 expression in response to heat shock at 42°C in A549 cells. |
 |  | Application Brief 1009: Hsp70 Loading Control NanoPro Assay | [show details] |
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The 70 kilodalton heat shock proteins (Hsp70) are a family of ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms. The Hsp70s are an important part of the cell's machinery, and help to protect proteins from misfolding under stress. We describe its use as loading control for EGF stimulation in HeLa and MCF10A cells. In the NanoPro assay, Hsp-70 is present as a single peak around pI 5.8 under the conditions described. |
 |  | Application Brief 1010: Caspase 3 NanoPro Assay | [show details] |
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Caspases 3 exists as an inactive proenzyme that undergoes proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. Its activation is an important apoptosis marker. The data shows increased signal with antibodies specific to the Caspase 3 p17 subunit in response to apoptosis induction through prolonged treatment of K562 cells with Iminatib (aka Gleevec®) at expected pI's around 6.3-6.5. |
 |  | Application Brief 1011: β-2-Microglobulin Loading Control NanoPro Assay | [show details] |
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β-2-Microglobulin, also known as B2M, is a component of MHC class I molecules, which are present on all nucleated cells. In the NanoPro assay, it presents a single peak around pI 6. The data show its application as a loading control for EGF stimulation in HeLa cells as well as MCF10A cells. |
 |  | Application Brief 1012: p-STAT3 NanoPro Assay | [show details] |
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The Signal Transducer and Activator of Transcription (STAT) proteins regulate many aspects of cell growth, survival and differentiation. The transcription factors in this family are activated by Janus Kinase (JAK). Dysregulation of this pathway is frequently observed in primary tumors and leads to increased angiogenesis, enhanced survival of tumors and immunosuppression. STAT3 is constitutively active in overexpressing BCR-ABL K562 myelogenous leukaemia cells. Imatinib (also known as Gleevec®), a BCR-ABL inhibitor, reduces STAT3 phosphorylation in these cells. STAT3 is also part of the Epidermal Growth Factor (EGF) signaling cascade as shown in MCF10A cells. |
 |  | Application Brief 1013: p-STAT5 NanoPro Assay | [show details] |
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The Signal Transducer and Activator of Transcription (STAT) proteins regulate many aspects of cell growth, survival and differentiation. The transcription factors in this family are activated by Janus Kinase (JAK). Dysregulation of this pathway is frequently observed in primary tumors and leads to increased angiogenesis, enhanced survival of tumors and immunosuppression. STAT5 is constitutively active in the overexpressing BCR-ABL K562 myelogenous leukaemia cell line. Imatinib (also known as Gleevec®), a BCR-ABL inhibitor, reduces STAT5 phosphorylation in these cells. STAT5 is also part of the Epidermal Growth Factor (EGF) signaling cascade as shown in MCF10A cells. |
 |  | Application Brief 1014: PLCγ1 NanoPro Assay | [show details] |
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Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) to produce the metabolite second messenger molecules inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Increase of IP3 results in elevated intracellular free Ca2+. PLC's are activated through G-protein coupled receptor stimulation as well as tyrosine receptor kinase activation and therefore bridge both important signaling pathways. The family of PLC's consists of 12 isoforms with different roles in signaling. For example, PLCγ1 forms a complex with activated EGF receptors, which leads to the phosphorylation of PLCγ at Tyr771, 783 and 1245. Here we detect the phosphorylation of PLCγ1 in HEK293 cells in response to EGF treatment. |
 |  | Application Brief 1015: ERK1/2 NanoPro Assay | [show details] |
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Extracellular signal-regulated kinases (ERK) or classical MAP kinases are widely expressed intracellular signaling molecules involved in regulation of meiosis, mitosis, and post-mitotic functions in differentiated cells. Many different stimuli (including growth factors, cytokines, virus infection, ligands for heterotrimeric G protein-coupled receptors, transforming agents, and carcinogens) activate the ERK pathway. We show an example of ERK phosphorylation in MCF10A cells in response to treatment with epidermal growth factor (EGF). |
 |  | Application Brief 1016: MEK1 NanoPro Assay | [show details] |
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Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and intracellular signals. While the functions of MEK1 and MEK2 are very similar, these kinases differ significantly in the way they are regulated. For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK1 activation in MCF10A cells treated with EGF. |
 |  | Application Brief 1017: MEK2 NanoPro Assay | [show details] |
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Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and intracellular signals. While the functions of MEK1 and MEK2 are very similar, theses kinases differ significantly in the way they are regulated. For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK2 activation in MCF10A cells in response to EGF stimulation. |
 |  | Application Brief 1018: Src NanoPro Assay | [show details] |
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Src is involved in regulating growth and differentiation in eukaryotic cells. Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. Phosphorylation of Tyr416 in the activation loop of the kinase domain by Csk upregulates enzyme activity, whereas phosphorylation of Tyr529 in the carboxy-terminal tail renders the enzyme less active. We evaluated Src response to EGF in A431 cells. |
 |  | Application Brief 1019: p-JNK NanoPro Assay | [show details] |
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c-Jun N-terminal kinases (JNK), originally identified as kinases that bind and phosphorylate c-Jun on Ser63 and Ser73, are mitogen-activated protein kinases which are responsive to stress stimuli, such as cytokines, UV-irradiation, heat shock, and osmotic shock, and are involved in T cell differentiation and apoptosis. JNK1, 2 and 3 share a total of 10 isoforms with pI's ranging from 5.4-7.6. All three JNK kinases share a similar Thr/Tyr phosphorylation site (T183/Y185). We evaluate change in JNK phosphorylation in UV-treated HEK293 cells and imatinib-treated K562 cells using a dual phospho-antibody against that site. |
 |  | Application Brief 1020: phospho-p27/Kip1 NanoPro Assay | [show details] |
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p27, also known as Kip1, is a cell cycle regulatory/inhibitory protein. It is similar to other members of the "Cip/Kip" family which includes the p21Cip1/Waf1 and p57Kip2 genes. p27 shares their functional characteristic of being able to bind several different classes of Cyclin and CDK molecules, acting as a CDK inhibitor. We show the response of p27 to EGF treatment in MCF10A cells. |
 |  | Application Brief 1021: 4E-BP1 NanoPro Assay | [show details] |
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Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been shown to potentially play a role in energy homeostasis. We show 4E-BP1 activation in MCF10A cells in response to EGF using total and anti-phospho 4E-BP1 antibodies that enable distinction between phospho and non-phospho peaks. |
 |  | Application Brief 1022: 4E-BP2 NanoPro Assay | [show details] |
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Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/AKT pathway and FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been shown to potentially play a role in energy homeostasis. We show 4E-BP2 activation in MCF10A cells in response to EGF and 4E-BP2 inhibition in MCF7 cells with LY294002 (PI3 kinase inhibitor). |
 |  | Application Brief 1023: Crk-L NanoPro Assay | [show details] |
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Crk-like protein (Crk-L) is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. It has been shown to activate the RAS and JUN kinase signaling pathways and transform fibroblasts in a RAS-dependent fashion. Crk-L is a substrate of the BCR-ABL tyrosine kinase and plays a role in fibroblast transformation by BCR-ABL. We show that Crk-L phosphorylation is reduced in response to Imatinib (commonly known as Gleevec®) treatment in K562 cells. |
 |  | Application Brief 1024: c-Myc Epitope Tag NanoPro Assay | [show details] |
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The c-Myc-tag
consists of a short peptide sequence (MEEQKLISEEDLLM). EGFP was expressed with a c-Myc-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed. |
 |  | Application Brief 1025: FLAG Epitope Tag NanoPro Assay | [show details] |
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The FLAG-tag
consists of a short peptide sequence (MADYKDDDDKM). EGFP was expressed with a FLAG-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed. |
 |  | Application Brief 1026: HA Epitope Tag NanoPro Assay | [show details] |
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The HA-tag consists of a short peptide sequence (MAYPYDVPDYASM). Here, EGFP was expressed with a HA-tag at both the C- and N-terminal of the protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed. |
 |  | Application Brief 1027: ASNS NanoPro Assay | [show details] |
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The enzyme-drug, L-asparaginase, has been used since the 1970s to treat acute lymphoblastic leukemia. Asparagine synthetase (ASNS) expression has been found to be correlated with L-asparaginase efficacy in leukemia cell lines, in leukemia primary tumor samples, and more recently, in cancer cell lines from other tissues of origin. Silencing ASNS expression by RNAi has indicated the L-asparaginase/ASNS relationship is causal and suggests that ASNS expression may be useful as a predictive clinical biomarker of L-asparaginase efficacy. ASNS presents as a single peak in the NanoPro assay. Expected changes of expression are observed upon siRNA as well as L-asparaginase
treatment. |
 |  | Application Brief 1028: LDH NanoPro Assay | [show details] |
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In many different species, lactate dehydrogenase (LDH) constitutes a major checkpoint of anaerobic glycolysis by catalyzing the reduction of pyruvate into lactate. LDH in its native form, is a tetramer of LDHA (calculated pI: 8.4) and LDHB (calculated pI: 5.7) proteins in different combinations. When the number of protein B over protein A increases, isozymes become more efficient in catalyzing the oxidation of lactate to pyruvate. LDH overexpression has been implicated in the pathogenesis and progression of many cancers and may constitute a valid therapeutic target for diseases. |
 |  | Application Brief 1029: cIAP1 NanoPro Assay | [show details] |
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cIAP1 is a member of the inhibitor of apoptosis (IAPs) family of proteins. It is up-regulated in several human cancers and plays an important role in tumor survival. cIAP1 functions to prevent cellular apoptosis by preventing the activation and/or inhibiting the function of different caspases. |
 |  | Application Brief 1030: GFP NanoPro Assay | [show details] |
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Green Fluorescent Protein (GFP) was originally isolated from jellyfish and exhibits a bright green fluorescence when exposed to blue light. In cell and molecular biology, GFP is commonly utilized as a reporter of protein expression. GFP can be introduced and its expression maintained in a wide range of cell lines and organisms. Thus, assays suitable for GFP detection offer useful tools for following the expression of proteins for which high affinity antibodies are lacking. The data demonstrates identification of a highly sensitive antibody for GFP detection via NanoPro assay. |
 |  | Application Brief 1031: eEF2 NanoPro Assay | [show details] |
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Eukaryotic elongation factor-2 (eEF2) catalyzes ribosome translocation during translation of mRNA. Phosphorylation of eEF2 by eEF2 kinase (also known as CaM kinase III) inactivates the protein and can block protein synthesis. Stimulation of growth is associated with a decrease in eEF2 phosphorylation. Inhibition of eEF2 phosphorylation in the hippocampus has recently been associated with an anti-depressant eect. We show that insulin treatment reduces phosphorylation of eEF2 in H4IIE cells. Inhibition of phosphorylation of eEF2 is restored in the presence of Nh125. |
 |  | Application Brief 1032: Mfn1 NanoPro Assay | [show details] |
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Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) are two highly homologous mitochondrial outer membrane proteins necessary for mitochondrial fusion. Mitochondrial fusion is necessary to maintain mitochondrial health and function, and mitochondrial dysfunction is implicated in diseases such as Charcot-Marie-Tooth 2A and dominant optic atrophy, as well as in multiple tissue types. Phosphorylation of these proteins has not been widely studied. Our data show detectoin of Mfn1 protein in three neuronal tissues: retina, optic nerve, and the superior colliculus region of the brain in a mouse model, DBA/2J, a widely accepted model for glaucoma. |
 |  | Application Brief 1033: Mfn2 NanoPro Assay | [show details] |
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Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) are two highly homologous mitochondrial outer membrane proteins necessary for mitochondrial fusion. Mitochondrial fusion is necessary to maintain mitochondrial health and function, and mitochondrial dysfunction is implicated in diseases such as Charcot-Marie-Tooth 2A and dominant optic atrophy, as well as in multiple tissue types. Phosphorylation of these proteins has not been widely studied. Our data show detectoin of Mfn2 protein in three neuronal tissues: retina, optic nerve, and the superior colliculus region of the brain in a mouse model, DBA/2J, a widely accepted model for glaucoma. |
 |  | Application Brief 1034: Survivin NanoPro Assay | [show details] |
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Survivin, an inhibitor of apoptosis protein (IAP) is a pro-survival molecule that is increased in nearly every human tumor studied. In head and neck squamous cell carcinoma, Survivin levels are significantly greater than in normal upper aerodigestive mucosa. High Survivin levels in these tissues correlate with a higher probability of nodal metastasis and loco-regional recurrence, but may also indicate higher radiosensitivity. Survivin includes multiple sites for post-translational modification, including 4-5 major phosphorylation sites. |
 |  | Application Brief 1035: Phospho-p38 NanoPro Assay | [show details] |
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The Mitogen Activated Protein Kinase p38 is activated by stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation and apoptosis. We show that Interleukin-1 (IL-1) treatment induces phosphorylation of p38 in fibroblasts. In addition, two independent antibodies recognize peaks at pIs 5.4 and 5.8 for phosphorylated p38. |
 |  | Application Brief 1036: GAPDH Loading Control NanoPro Assay | [show details] |
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GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) is a housekeeping gene found in most tissues and cells. Because GAPDH expression is fairly constant in a variety of tissue and cell types, this protein is often used as a control in comparisons of protein expression levels. Here, we describe its use as a loading control for cIAP1 inhibition in PBMCs. In the NanoPro assay, GAPDH is present with a major peak around pI 8.9 under the conditions described. We also demonstrate the multiplexing capabilities of the NanoPro assay and the detection of cIAP1 and GAPDH in the same capillary. |
 |  | Application Brief 1037: PTEN NanoPro Assay | [show details] |
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Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor protein that is frequently mutated or deleted in many tumors. PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PtdInsP3) inhibiting phosphoinositide 3-kinase (PI3K) activation of AKT repressing cell growth, proliferation and survival. Recent studies show that PTEN phosphorylation alters its stability and function. |
 |  | Application Brief 1038: Histone NanoPro Assay | [show details] |
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Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11). |
 |  | Application Brief 1039: Acetylation NanoPro Assay | [show details] |
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Acetylation is a common post-translation modification typically observed on chromatin proteins as well as other regulatory proteins and metabolic enzymes. The reaction is catalyzed by N-terminal acetyltransferases, occurs predominantly during protein synthesis and appears to be irreversible. Pan-Ac-Lysine antibodies are widely used for detection of acetylation of imunoprecipitated proteins when no specific antibodies are available. |
 |  | Concentrating on AAV Impurities with Ultrasensitive Total Protein Detection on Simple Western | [show details] |
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In this Application Note, we show how to achieve ultrasensitive total protein detection using the 5X biotin labeling reagent with a focus on AAV analysis. |
 |  | Automated Profiling of PROTAC®-Induced Cereblon Neosubstrate Degradation Using Simple Western | [show details] |
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Here, we present data showing the power of using automated Simple Western platforms to screen panels of Degrader and IMiD compounds in order to quantify degradation activity. In this study, we demonstrate the time savings achieved by automating these large screens as well as Simple Western’s ability to accurately quantify DC50 and Dmax values for specific Degraders and IMiDs. |
 |  | A Multi-Antigen Serology Assay for COVID-19 using Simple Western | [show details] |
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A multi-antigen serology assay for COVID-19 using Simple Western furthers understanding of the COVID-19 disease progression and the efficacy of a vaccine. |
 |  | Simple Western Assays for Detection of ACE2 and TMPRSS2, Key Players in SARS-CoV-2 Infection | [show details] |
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In this Application Note, we demonstrate how Simple Western can be applied for the detection and characterization of ACE2 and TMPRSS2, key players in SARS-CoV-2 infection. As an open platform, any commercial or custom antibody may be used for target protein detection on Simple Western. Here, we used an anti-ACE2 antibody from R&D Systems and an anti-TMPRSS2 antibody from Novus Biologicals to develop immunodetection assays for these proteins in purified form and expressed in human cells. This resulted in assays that were highly sensitive over a large dynamic range, providing molecular weight information and even quantification in a human cells. |
 |  | Reprobe Your Immunoassay Samples Using RePlex with Jess | [show details] |
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To gain the most data out of your precious sample, Jess provides a two-step immunoassay that is
performed within the same capillary. This new feature, called RePlex removes the antibodies from the
first round of probing to perform either a second round of probing with new antibodies or total protein
detection. Importantly, RePlex efficiently removes antibodies between probing without compromising
the integrity of the immobilized protein or its epitopes, allowing for excellent reproducibility across
cycles. With Jess’s chemiluminescence and fluorescence channels, you can detect multiple targets per
cycle. The second cycle can also be dedicated to total protein detection so that you can normalize your
data with confidence. All the steps of RePlex are automatically performed with Jess, providing more data,
and lowering the cost of reagents and consumables per sample. |
 |  | Wes and Milo Synergize to Profile Immune Cell Populations in the Tumor microenvironment- Application Note | [show details] |
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In this application note, we’ll show you how Wes™ and Milo™ partner to get you critical
answers to 1) what type of immune cell populations are present in a sample and then 2) what percentage of cells in that sample
make up a specific immune cell subtype. |
 |  | Simple Western Analysis of Adeno-Associated Virus (AAV) Proteins for Cell and Gene Therapy | [show details] |
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In this application note, you’ll see how CGTC has used highly-specific antibodies exclusively manufactured by PROGEN with fully automated Simple Western™ assays on Wes™ to monitor and characterize AAV capsids during product purification. |
 |  | Wes and Milo Tumor Microenvironment App Note Japanese | |
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 |  | Wes and Milo Synergize to Profile Immune Cell Populations in the Tumor Microenvironment | [show details] |
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Immunotherapy has opened the door to a new era in cancer treatment where dramatically successful and durable responses are being reported. Still, the population of patients who respond are a minority, and this highlights the need for a deeper understanding of the immune response to cancer. To that end, accumulating evidence suggests that the heterogeneous makeup of the tumor microenvironment (TME) is a major contributor to the discrepancies in response observed. |
 |  | Multiplexed Western Blotting Redefined: Superplexing on Jess | [show details] |
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In this application note, we simultaneously assess p-Akt and total Akt levels using different detection channels combined with in-capillary protein normalization to demonstrate the superplexing power Jess possesses. |
 |  | Using the Simple Western Total Protein Assay to Normalize Immunoassay Data in the Same Run | Japanese | [show details] |
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シンプルウェスタンTotal Protein Assayの同時測定で、イムノアッセイデータのノーマライゼーション |
 |  | High-Throughput Glycan Characterization using Simple Western | Japanese | [show details] |
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シンプルウェスタンを用いてハイスループットで 糖鎖のキャラクタリゼーション |
 |  | Bioprocess Contaminant Detection using Simple Western | Japanese | [show details] |
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Simple Westernでバイオプロセス中の不純物質の検出
医薬品製造過程関連の不純物の同定および定量 |
 |  | Easy Transfer of Your Traditional Western Blot to Wes | Japanese | |
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 |  | High-throughput glycan characterization using Simple Western | |
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 |  | Bioprocess Contaminant Detection using Simple Western | [show details] |
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In this application note, we focus on the accurate detection of four candidate contaminants that may be present during various stages of the therapeutic protein and vaccine development processes: host cell protein (HCP), Protein A, green fluorescence protein (GFP) and bovine serum albumin (BSA). |
 |  | Monitoring Target Engagement in Drug Discovery: Application of Wes to the Cellular Thermal Shift Assay | [show details] |
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In drug discovery, confirmation of in-cell target engagement is a critical component of the drug development process. Confirming that a drug candidate engages its proposed target in the cell and determining the concentration at which it exerts the desired effect(s) fulfill fundamental criteria for translation to activity and efficacy in its target tissue. Thermal shift assays (TSA) are regularly used by industry and academia to uncover or confirm interactions using purified proteins. Recently, this type of assay has been adapted to a cellular format and is called the cellular thermal shift assay (CETSA). In this application note, quantitative and reproducible CETSA data generated with ProteinSimple’s Wes instrument (CETSA-Wes) are presented. This assay verifies drug target binding, and given its quantitative nature, the half maximal inhibitory concentration (IC50) is also calculated. |
 |  | Using the Simple Western Total Protein Assay to Normalize Immunoassay Data in the Same Run | [show details] |
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In this how-to-guide, we'll show you how to use the Total Protein Detection Module (DM-TP01) with any immunoassay detection module of your choosing to get total protein and immunoassay data in the same run. |
 |  | Accelerated Serum Biomarker Verification and Validation with Wes and Ella | [show details] |
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In this application note, we’re honing in on the biomarker verification and validation steps. Proof-of-concept data was generated to demonstrate how Simple Western and Simple Plex assays data give similar trends and work together to give you fast, sensitive, and precise information about your biomarkers of interest. |
 |  | Multiplexing with Simple Western | |
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 |  | Simple Western Streamlines Serum Antibody Analysis | [show details] |
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Scientists measure serum antibody levels to confirm immune responses against a bacteria to diagnose infections, to test for antibody production after vaccination, and to detect the presence of autoantibodies in autoimmune diseases. Traditional Western blots are often used to detect these antibodies, but testing with Western blots means a lot of hands-on time. After the antigen of interest is separated by SDS-PAGE and transferred to a membrane, each lane has to be cut into individual strips so patient serum samples can be individually tested for the presence of specific antibodies. Then you have to process and analyze them manually.
Simple Western assays happen in individual capillaries, and everything from sample separation to data analysis is completely automated. No more cutting individual strips, washing and incubating them, or lining them all up with a molecular weight marker before detection. Just pipette your sample into the wells of your assay plate, set up your run, and you're done! And all that manual data analysis is gone too — Compass for Simple Western does it all for you. Did we mention you only need 10 µL of diluted serum per data point? That means you'll get a lot more data points for every 1 µL of neat serum.
In this application note, we used Simple Western to detect autoantibodies in lupus patient serum as a model system to generate proof-of-concept data for the assay. But you can use this method any time you need to detect and quantitate specific serum antibodies. In fact, check out how researchers are using this assay to detect Salmonella antibodies without having to cut blots into individual strips. |
 |  | Breaking Laser Capture Microdissection Sample Size Road Blocks with Simple Western | [show details] |
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Laser capture microdissection (LCM) is a powerful tool to identify and isolate a pure sample of the cell type you're
interested in. But, proteomic studies with LCM samples are really restricted by the small amounts of tissue collected with
each capture since there just isn't much to work with. You often have to use the entire sample captured for traditional
Western blot analysis and that only leaves you with one data point! So, researchers often use 2D Electrophoresis and
mass spectrometry instead to max out the amount of data they can generate. Both of these methods have their own
limitations when it comes to ease-of-use and reproducibility though. Immunohistochemistry is also used to analyze LCM
samples as it's a more accessible technique, but it really doesn't give you a lot of info either.
Simple Western is an automated capillary-based immunoassay that changes the proteomic research game. You only
need 1-10 µL of LCM sample for each data point, so you'll get more data points for each sample you collect. Not to
mention the sensitivity that comes with it will even let you analyze proteins you couldn't previously do with traditional
Western blot. And it's all wrapped up in a simple workflow that minimizes your hands-on time. Simple Western is a
sensitive, easy-to-use analytical tool that ups the ante on protein analysis with LCM samples.
In this application note, we'll show you two examples of how Simple Western changed what researchers could do with
their precious LCM samples for the better |
 |  | High Molecular Weight Protein Analysis Made Simple | |
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 |  | Total Protein Analysis the Simple Western Way | [show details] |
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The Simple Western Immunoassay is the gel-free, blot-free and hands-free solution for researchers looking for a better way to get their Western blot data. The simple fact that you get analyzed data in just three hours with only 30 minutes of hands-on time changes things forever! |
 |  | Peggy: size- or charge-based Western blotting at the
push of a button | [show details] |
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Peggy enables researchers to follow up a size-based immunoassay with a charge-based assay on one platform using the same sample. The charge-based analysis provides an information-rich, complementary data set, elucidating ratios of protein variants and providing leads for biomarker development. Like other Simple Western products, Peggy provides a fully automated solution, from loading samples
all the way to peak analysis. |
 |  | Easy Transfer of Your Traditional Western Blot to Wes | [show details] |
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Wes takes the benefits of Simple Western assays a step further by simplifying their workflow with pre-filled plates and disposable capillary cartridges. This application note gives an overview of how to transfer a traditional Western blot assay to Wes. |
 |  | Detailed Characterization of ERK1 and ERK2 Phosphorylation | [show details] |
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Monophospho- and diphospho-ERK isoforms are not resolved by traditional Western blot analysis, and the sample quantity required is relatively large. The Firefly ERK1/ERK2 assay can distinguish and quantify unphosphorylated, mono and dual-phosphorylated isoforms of ERK1 and ERK2, allowing a more accurate determination of ERK activation than Western blots provide. |
 |  | Screening siRNA and Verifying shRNA Knockouts | [show details] |
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RNA interference (RNAi) is an RNA-dependent gene silencing mechanism that can affect the expression of specific genes by inhibiting translation or suppressing transcription epigenetically. Using Firefly assays, RNAi effects such as impact on phosphorylation or silencing can be studied functionally in samples as small as 100 cells. An additional benefit of the small samples size is that a variety of conditions can be studies in a single assay. |
 |  | Unraveling Neurodegenerative Diseases with Jess | [show details] |
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Over the course of Dr. Nicolas Bazan’s several decades-long career, he has been a research scientist, teacher, mentor, community leader, author, screenplay co-writer, executive movie producer, patron of the arts, and entrepreneur. Dr. Bazan, who has published close to 500 peer-reviewed publications, sings the praises of Simple Western assays with Jess. Jess has dramatically improved the protein analytical methods on which his laboratory relies to study the cellular and molecular events of neurodegenerative disease onset and progression. |
 |  | Conducting Novel Therapeutics Research of Human Gliomas with Jess | [show details] |
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Learn how Jess furthers therapeutic research of human gliomas. Reduce preparation time while saving reagents, samples, and antibodies with target optimization. |
 |  | Jess Breaks New Quantitative Ground in New Chemical Entities Screening at TCG Lifesciences | [show details] |
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Dr. Sarkar from TCG Lifesciences uses Jess, the automated Western blotting platform from ProteinSimple, for high throughput screening of new chemical entities (NCE) giving them confidence in the data. |
 |  | PROTAC Studies Take Off in Drug Discovery, and Jess is On Board | [show details] |
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Aurelia Bioscience is solving drug discovery challenges by taking on PROTAC studies with ProteinSimple’s Jess and Simple Western assays. |
 |  | Rebel without a Cause - Searching for the Origins of Parkinson's Disease with Wes | [show details] |
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ProteinSimple supports Parkinson’s disease research. See how Wes does Simple Western protein analysis on Parkinson's disease samples. |
 |  | Simple Western and the Race to Develop a Vaccine for the COVID-19 Coronavirus | [show details] |
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As the world finds itself in the middle of the worst pandemic in a century, finding a vaccine to the deadly COVID-19 coronavirus could not be more urgent. Dr. Ferrari and his colleagues at DIOSynVax (Digitally designed, Immune Optimised Selected and Synthesized Vaccines) are working to
develop a DNA vaccine, and Simple Western from ProteinSimple is an important part of that process. |
 |  | Wes Validates the Potential of RNA-Based Metabolic Therapies at MiNA Therapeutics | [show details] |
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“I can run immunoassays on Wes for an entire animal experiment in a single day. This is something I wouldn’t even try with traditional Western blots.”
Jon Voutila, Ph.D., Senior Research Scientist, MiNA Therapeutics, London, UK |
 |  | Wes Zips Through Pharmacodynamic Biomarker Discovery Projects at the University of Glasgow | [show details] |
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“I was able to screen various antibodies in a single run within half a day. This had previously taken weeks and months using traditional Western blotting methods.” |
 |  | Quantifying Protein Phosphorylation in Platelets with Wes | [show details] |
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“[Wes] Provides quantitative data, saves time and uses far less sample.” |
 |  | Lower Limit of Detection Advances Understanding of Metabolic Signaling in Human Muscle | [show details] |
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"Wes allows us to measure cell signaling proteins from a single muscle fiber segment by lowering our limit of detection nearly ~20-fold from Western blotting. It also allows for much more precise, reproducible, and faster results to be generated, in addition to far greater ease of operation, analysis, and waste disposal." |
 |  | Detecting Low Abundance Proteins in Precious Pediatric Cancer Samples with Wes | [show details] |
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"Some of the proteins we wanted to detect are low in abundance and it's really hard to get a good amount of protein for traditional Western blot. The low protein concentrations required by Wes makes it easier to save precious samples ensuring protein detection." |
 |  | Wes Gives Researchers Fast Answers at Cancer Research UK, Cambridge Institute | [show details] |
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“Wes is easy to use, convenient, and gives answers quickly. The throughput and system automation is a definite advantage.”
— Jane Gray, Ph.D., Head of Research Instrumentation, Cancer Research UK Cambridge Institute |
 |  | Wes Speeds Up the Core at Xavier University of Louisiana | [show details] |
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“We’ve been able to increase productivity dramatically with Wes. The results are much better too, given that it removes the blotting step all together.”
— Melyssa Bratton, Ph.D., Manager of the Cell, Molecular, and Biostatistics Core, Xavier University of Louisiana |
 |  | Getting to Better Outcomes for Osteoarthritis Patients with Wes at the University of Chester | [show details] |
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"Wes gives me more throughput so I can get more replicates and trust my results a little bit more, and I can trust them straight away rather than having to do a lot of repeats. And obviously I like getting results the same day rather than having to wait two days."
— Emma Wilson (née Humphrey), Ph.D., Lecturer in Molecular Biology, Institute of Medicine, University of Chester |
 |  | Making the Link Between Obesity and Cancer with the NanoPro 1000 at Roseman University of Health Sciences | [show details] |
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"The Simple Western Charge assay, a capillary isoelectric focusing immunoassay, exceeded the reliability of 2D Western blots for resolving recombinant PKG-Iα and PKG-Iβ and uses 100,000X less sample quantity, making it well-suited for clinical disease proteomics because protein isoforms and post-translational modifications can be detected in precious tissue biopsies."
— Mary G. Johlfs, M.S., Director of Research Operations/Scientist, Roseman University of Health Sciences |
 |  | Wes Takes on Far-Western Blotting to Detect Protein Phosphorylation at UConn Health | [show details] |
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"The no-gel, no-transfer, no-membrane features are beneficial for the reproducibility of the assay. I really like this system over traditional Western."
— Kazuya Machida, M.D., Ph.D., Associate Professor, Department of Genetics and Genome Sciences, UConn Health |
 |  | Wes is Keeping Things Green at the University of Alberta | [show details] |
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“The traditional blotting method can use up to 300 pieces of plastic and Wes uses two. What’s even better is that Wes can run up to 40 proteins in three days. It would take about a week to run 40 proteins using Western blotting.”
— Jamie Boisvenue, Cardiovascular Research Technician, Department of Pediatrics, University of Alberta Faculty of Medicine & Dentistry |
 |  | Wes Cuts Time to Results and Solves the Membrane Protein Challenge at the University of Nevada School of Medicine | [show details] |
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"We had been trying to study receptor proteins with traditional Westerns but it was frustrating — we just could not get it. A few months with Wes and we've already had way more success than we had in three years."
— Brian Perrino, Ph.D., Associate Professor, Department of Physiology and Cell Biology, University of Nevada School of Medicine |
 |  | Wes sets a new pace for Alzheimer‘s research at the University of Texas Health Science Center | [show details] |
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"The rapid, reproducible results on small amounts of tissue/cell lysates have allowed me to generate a more thorough data set on additional proteins, samples, conditions and brain regions all in the same amount, or even less time, as traditional Western blot. This technology has made it possible for small research labs to compete with the pace that research is conducted in large labs or companies."
— Miranda Orr, Ph.D., Postdoctoral Fellow, University of Texas Health Science Center |
 |  | Wes Ups the Data Points Count on Laser Capture Microdissection Samples at East Tennessee State University | [show details] |
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”We collect samples from laser microdissection and were using our entire sample on just one regular Western blot. With Wes, we can do multiple assays with one sample collection.“
— Mary Howell, Laboratory Coordinator, Department of Internal Medicine, East Tennessee State University |
 |  | Wes changes the brain receptor research
game at the University of Texas | |
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 |  | In-Capillary Immunoassay and Total Protein Detection for Adeno-Associated Virus (AAV) Proteins During Purification from Whole-Cell Lysate -- Dr. Chris Heger | [show details] |
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During adeno-associated virus (AAV) manufacture, critical quality attributes must be monitored including the presence, identity, and purity of viral vector proteins. Traditionally, the identity and purity of these proteins is monitored by Western blot using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS-PAGE is notoriously challenging and labor-intensive. Additionally, AAVs are limited in sample size as they are difficult to manufacture, and demand outstrips supply. Here, we have developed a method to monitor the purification of AAV2 using automated capillary electrophoresis followed by immunoassay and total protein detection directly in the capillary, eliminating the need for SDS-PAGE. AAV2 was purified from HEK293 cell lysate using affinity chromatography, and the steps of the purification process (load, flow-through, wash and elution) were monitored by the capillary-based immunoassay. VP1, VP2 and VP3 capsid proteins were resolved and identified either individually or simultaneously, depending on the AAV antibody used for detection, and the total protein assay monitored the presence of impurities. The sensitivity of this assay reduced sample size down to 3 µL of sample, corresponding to approximately 400 pg or 1x108 genomic copies loaded per well. We anticipate that this in-capillary immunoassay and total protein detection can replace traditional SDS-PAGE methods in AAV manufacturing workflows. |
 |  | Detection of MISEV recommended EV Protein-Markers using Automated Western Blotting -- Lisa Meyer | [show details] |
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The limited amount of material and the diverse methods for isolation of extracellular vesicles (EV) pose unique challenges to proper characterization of experimental EV preparations. The “Minimal Information for Studies of Extracellular Vesicles” (MISEV) guidelines recommend characterizing preparations for both trans-membrane-, cytosolic- and contaminating non-EV proteins. However, compliance with these guidelines can be a considerable effort due to lack of easy and robust analytical protocols and the time consuming and user variable nature of standard western blotting protocols. Here we present a simple method for isolation of EVs and a simple western blotting platform for automated protein separation and immunodetection of MISEV-recommended proteins. The total EVs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 mL plasma or 2-20 mL urine, respectively. Intact vesicles were eluted and the EV-depleted biofluid fraction was collected from the flow-through. A small fraction (4 μL) was analyzed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of MISEV-recommended proteins. A range of specific antibodies were identified and the EV fractions were shown to be enriched in EV-proteins, whereas contaminating non-EV proteins were significantly reduced. Isolation of EVs was necessary to allow detection of the low abundant EV protein markers, whereas non-EV proteins were readily detectable both in the neat biofluids and in the EV-depleted flow-through. We characterized the effect of washing on the purity of EV isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine EV isolations. In conclusion, Simple western blotting protocols were established for quality control of isolated EVs in accordance with MISEV-guidelines. EVs isolated using affinity-membrane spin columns were shown to be enriched in EV markers and depleted for non-EV proteins. |
 |  | AACR Dermody Poster featuring Jess - Japanese | |
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 |  | Determination of antibody’s specificity towards phosphorylated protein targets with automated in-capillary enzyme treatment and immunoassay -- Dr. Daryl Taketa | [show details] |
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Protein phosphorylation is a reversible reaction that is integral in numerous signaling cascades. Characterization of signaling cascades has been largely detected by immunoblotting with phospho-specific antibodies, which may or may not have enough specificity or affinity. Currently, a separate lysate without any phosphatase inhibitors or a separate blot is needed to determine an antibody’s specificity. Here we describe a simple assay that leverages automation and quantitation with capillary electrophoresis-based immunoassay (CEIA) to assess the specificity of these antibodies with a single lysate preparation. In this study, three lysate models are used: K562 ± TNFα treatment, 50 ng/mL phorbol myristate acetate (PMA) differentiated THP-1 ± 1 μg/mL lipopolysaccharide (LPS) treatment, and cytotoxic T lymphocytes (CTL) ± 10 ng/mL PMA and 500 ng/mL ionomycin treatment. K562 cell lysates are commercially purchased whereas THP-1 lysates are generated in-house. For CTL cells, whole blood cells from a single donor are isolated and expanded with commercially available kits. Expanded CTL cells are then stimulated with PMA and ionomycin for 15 minutes. Untreated and treated lysate samples are separated and captured to the inner lumen of the capillary wall with UV activated crosslink chemistry. Cross-linked proteins are treated with lambda phosphatase for 1 hour followed by the immunoassay to investigate the specificity of antibodies against phosphorylated protein targets respective to each activated pathway using either chemiluminescent or fluorescent detection. Preliminary data suggest phospho-specific signal decreased >90% with no significant changes to the non-specific noise. The method described here eliminates the need for multiple lysate preparations or an additional blot to assess an antibody’s specificity to a phosphorylated protein target. |
 |  | Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K/AKT pathway proteins -- Dr. Jessica Dermody | [show details] |
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The P13K/Akt signaling pathway modulates growth, survival and apoptosis and this pathway is frequently modulated in human cancers contributing to resistance to radiation and chemotherapy treatments. Akt is a target for specific inhibition and recently, a number of small molecules have been developed the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, Pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2 and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of Replex™ to characterize the P13K/Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immune assays or immunoassay with total protein on the Simple Western platform chemiluminescense detection. Assays with control and LY294002 inhibitor- treated samples were developed. Proteins were first separated based on molecular weight based on capillary electrophoresis, followed by immobilization via UV-crosslinking. Next P13K/Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe was removed with the detection probe with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading and/or other systemic errors. These results present the utility of the RePlexTM to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.
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 |  | Profiling immune cell populations in the tumor microenvironment with complementary capillary-based and single-cell Western assays -- Dr. Charles Haitjema | [show details] |
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The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell Western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell Western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like Western blot and flow cytometry. |
 |  | Multiplexed Protein and RNA Quantification on a Single Instrument Harmonizes Multi-omic Analyses of Biomarkers for Immunotherapies and Targeted Therapies in Non-Small Cell Lung Cancer -- Dr. Chris Heger | [show details] |
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Multi-omic approaches can combine protein, DNA, and RNA analyses to elucidate diagnostic biomarkers and pathways, advancing our understanding of complex diseases. These assays, however, require different technologies and platforms to resolve the distinct physico-chemistries of protein and DNA/RNA. In contrast, single-platform quantification of proteins and nucleic acid markers offers many potential benefits, including reduced sample requirements, decreased inter-assay variability, streamlined and less error-prone workflows, and integrated results reporting. Here we demonstrate expanded capabilities of an established protein analysis system (Simple Western, ProteinSimple®) to characterize nucleic acids, and show that this system can quantify oncogenic tyrosine kinases, immune checkpoint proteins, RNA translocations, and other mRNA transcripts associated with targeted or immune-based therapies for non-small cell lung cancer (NSCLC). |
 |  | Poster MRC Dystrophin quantification - Film, LICOR Odissey and Wes Japanese | |
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 |  | A Single-Platform Technology for Proteogenetic Biomarker Analysis in Oncology: Complementary Protein and RNA Quantification Relevant to Targeted and Immuno-therapies in Non-Small Cell Lung Cancer | |
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 |  | Maternal Obesity Reduces Skeletal Muscle Insulin Sensitivity in Fetal and Juvenile Japanese Macaques | |
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 |  | Precision Cut Cancer Tissue slices as human model for the testing of immune-modulatory compounds | [show details] |
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The goal of personalized medicine is to stratify individual patients to the appropriate treatment. This approach depends on extensive characterization of individual tumors and their sensitivity to therapeutics. In the context of the immunotherapy of cancer, information on the localization, abundance and activation of immune cells within individual tumors gained in importance. Here we present a preclinical drug testing model to monitor individual drug responses of patients to targeted immunotherapy with the checkpoint inhibitor Nivolumab (anti- PD-1) and subsequent applications. In this study, we were able to determine different populations of infiltrating immune cells within viable tumors from colorectal cancer patients using our drug testing platform and a variety of subsequent applications.
Analysis of immune cells was conducted on disaggregated cells from viable tumor slices. Disaggregation of precision cut cancer tissue slices was performed using the GentleMACS from Miltenyi. Immune cell subsets were analyzed by flow cytometric multiplexing of CD3, CD4, CD8 and CD45. Furthermore, we identified PD-1 positive cells among the CD45+/CD3+ lymphocyte population, indicating relevance for anti-PD-1 targeted therapy in colorectal cancer. Presence and localization of immune cells (CD45+) was confirmed by immunohistochemistry of tumor tissue slices. In addition, protein expression of CD8 and PD-1 was analyzed by Simple Western Size analyses. Cytokine secretion affected by Nivolumab treatment was analyzed in supernatants of tissue cultures using the proinflammatory panel from Meso Scale Discovery. The results demonstrated that immune cell compositions were stable and uniform within our precision cut cancer tissue slices both pre- and post-cultivation, and pre- and post-treatment with Nivolumab. |
 |  | Quantification of Cyclin D1, Cyclin D2, Ikaros, Aiolos and Cereblon Proteins by Capillary Immunoassay in Patients With Newly Diagnosed Multiple Myeloma and Analysis of Their Impact on Patient Survival | [show details] |
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Multiple myeloma has been comprehensively analyzed using high-throughput genomic technologies. Although a large number of biomarkers have been described, most of them were not subsequently validated at the protein level. In fact, the unresolved difficulties in studying the proteome have made the quantification of messenger RNA (mRNA) an indirect measure of protein expression. However, many studies have shown that levels of mRNA cannot be used as surrogates for protein levels. The amount of myeloma cells obtained after purification of patient samples is usually very limited, which precludes the possibility of quantify protein levels using standard Western Blot analysis. |
 |  | Analysis of minute amounts of clinical biospecimen for the AXL receptor expression using real-time PCR and Simple Western Size technology. | [show details] |
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AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and has been revealed as the most highly expressed gene in preclinical models with acquired resistance, and second most common alteration in EGFR (epidermal growth factor receptor) inhibitor-resistant tumors, behind the T790M mutation. It has become obvious that targeted therapy of patients has to be monitored by taking and analyzing biopsies on a regular basis. Therefore, laboratory methods have to be adapted. |
 |  | Characterization of a biological active soluble Human Cytomegalovirus gH/gL/pUL128/130/131 pentameric complex | [show details] |
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Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of non-genetic birth defects, and development of a prophylactic vaccine against HCMV is a top priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during vaccine manufacturing process. |
 |  | Predicting response to Mcl-1 targeting agents in Non-Small Cell Lung Cancer (NSCLC) and Multiple Myeloma (MM) cell lines. | [show details] |
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Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis. |
 |  | Deleterious effects of amyloid beta peptide in the neuromuscular junction: consequences in ALS disease. | [show details] |
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Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of adults which preferentially attacks the neuromotor system. It has been shown that Amyloid-beta (Aβ) levels are elevated in spinal cords of late-stage superoxide dismutase 1 (SOD1) G93A mice (model of familial amyotrophic lateral sclerosis [ALS]) and that Aβ peptide(s) were localized predominantly within affected motor neurons (MN) and surrounding glial cells. Moreover, neuromuscular junction (NMJ) loss and MN degeneration were reduced in SOD1 mice when APP was genetically ablated, suggesting that endogenous APP actively contributes to the pathophysiology of this form of ALS.
Additionally, Aβ and glutamate have been physiologically found in NMJs. Previous work done in our lab, showed the tight relationship between glutamate and Aβ in the NMJ. We showed that an interconnection between glutamate and Aβ peptide, as demonstrated in cortical and hippocampal neurons, is also operating in nerve-muscle co-cultures (Combes et al., 2015).
Here, using a nerve-muscle co-culture system, we studied the toxicity of Aβ and the mechanisms involved in the process of NMJ death. The aim of this study was to investigated the role and the mechanism of Aβ on an in vitro model of functional NMJ. |
 |  | Development of a IRAK1 Quantitative Protein Degradation Assay for In-vitro and Ex-vivo Target Engagement Using ProteinSimple Capillary Western Technology | [show details] |
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IRAK1 is a kinase which has been identified as a key regulator of cytokine signaling and is known to be involved in innate immune responses. Overexpression of these pleotropic cytokines have been implicated in various autoimmune diseases. Antagonists have shown clinical efficacy via modulation of various pro-inflammatory cytokine signals in various mouse models including the collagen induced arthritis (CIA) model. The identified project need was to demonstrate the engagement of IRAK1 with in-house chemical entities in-vitro by generating IC50s and ex-vivo using mouse tissue from the CIA model. To achieve this goal, a quantitative Capillary Western protein degradation assay was developed. |
 |  | Toxic events of beta-amyloid oligomers on cortical neurons and protective effect of beta-Estradiol: A mechanistic study (Neuro-Sys, SfN 2014) | [show details] |
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Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of beta amyloid peptide (Ab) has now shifted from insoluble Ab fibrils to smaller, soluble oligomeric Ab aggregates (AβO). Many evidences suggest that the neurodegenerative process would be due to the interaction of AβO with binding targets, activation of stress kinases, hyperphosphorylation of tau protein, caspase activation, loss of synapse, neuronal death, loss of cholinergic function, generation of reactive intermediates of oxygen (oxidative stress), or glutamate excitotoxicity. Urgent need for efficient new therapies is high, but could only be successful with an extensively comprehension of AβO degeneration process. In the present work, based on an in vitro primary cell culture treated with AβO preparation, we have carefully studied the cytopathological effects of AβO on neuronal death and then we have investigated the effect of 17-beta Estradiol (β-estr) on the degeneration process induced by AβO. Briefly we used rat cortical neurons (from E15). The cells were seeded in 96-well plates and intoxicated with AβO solution after 11 days of culture for 24 hours. β-estr was used at 100 nM (final concentration) and was added as pretreatment (1h before injuries). A co-incubation with selective inhibitors was performed for the mechanistic study. In parallel, western blotting (WB) analysis was done to quantify protein levels and their activation. We showed that β-estr was able to significantly protect neurons as well as glial cells from degeneration decreasing the caspase 3 activation and the massive mitochondrial stress (induced by AβO). Preservation of neurite network and synapsis integrity was also observed. Moreover, the large hyperphosphorylation of tau protein induced by AβO was significantly reduced with β-Estr. A mechanistic study was also performed co-incubating inhibitors of main survival pathways to try to better understand the mode of action of β-estr and the pathway involved in the AβO toxicity. We showed that the effects of -Estr were fully abolished blocking the MEK pathway as well as the DNA repair pathway (PARP-g) or the mitochondrial anti-apoptotic pathway (Bcl2). Interestingly the effect was inexisting coincubating β-estr with TrK receptor or Ras/Raf inhibitors showing the predominant role of growth factors paythway in its neuroprotective effect. Finally, we showed a large inactivation of AKT protein in presence of AβO that was reversed even over activated in presence of β-Estr. |
 |  | Eliminating Western Blot Variability for High Molecular Proteins with Sally Sue (HUPO 2014) | |
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 |  | Accurate Quantitation of Proteins Involved In Autoimmune Disease Using Simple Western (AAI & PEGS 2014) | |
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 |  | Accurate Quantitation of Recombinant Proteins with Simple Western (PepTalk 2014) | |
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 |  | Effects of antibody-mediated EGF-receptor inhibition on ERK1/2 isoform phosphorylation in organoid cultures (Indivumed, AACR 2012) | [show details] |
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Targeted anti-cancer therapy using small molecules or therapeutic antibodies is important to improve the treatment options of individual cancer patients whose tumor show specific expression patterns of respective target proteins. In order to enhance the development of new targeted drugs, novel and highly predictive in vitro drug testing models are needed which closely reflect the characteristics of each individual tumor.
Towards this end, Indivumed has developed a preclinical drug testing platform based on freshly cultivated tumor tissue slices which enables a detailed investigation of functional effects of classical chemotherapeutic drugs, small molecules and therapeutic antibodies in a natural tumor microenvironment. In addition, this multifunctional in vitro model permits the evaluation of target expression and analysis of signaling pathway activities.
The aim of the present study was to analyze and verify the functionality of an anti-EGFR antibody in colorectal cancer tissue slices using our recently developed drug testing platform. As readout of treatment effects changes in the expression and phosphorylation status of selected signaling proteins from two EGFR-related downstream pathways, the MAPK and Akt pathways, were evaluated by Meso Scale Discovery (MSD) assays and immunohistochemistry. To further analyze the complex regulation of phosphorylation pattern in more detail, we integrated the new NanoPro 1000 technology in our pathway analysis, enabling the identification of distinct isoform phosphorylations. This approach should help to extend the knowledge about individual drug responses among patients to further advance
personalized medicine. |
 |  | Development of a Robust Nanoimmunoassay and Immunohistochemical Assay for ASNS (MD Anderson, AACR 2012) | [show details] |
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The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly negatively correlated with L-ASP anticancer activity in ovarian cancer cell lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. However, quantitatively robust, single-antibody assays for ASNS expression have been absent from the literature. We therefore used a capillary-based isoelectric focusing (IEF) platform (the NanoPro 1000) to screen twelve ASNS antibodies for their specificity and sensitivity. Only two antibodies exhibited completely on-target activity (as shown by signal ablation by ASNS siRNA) and sufficient sensitivity. The on-target activity corresponded to a single band on Western blot and a single peak on the NanoPro 1000, suggesting the existence of just one ASNS protein isoform. Optimized, final NanoPro assay conditions yielded less than 8% CV, a 160-fold dynamic range, and Z′-factor of 0.82, indicating a robust assay that is amenable to high-throughput screening. We next used the best ASNS antibody to develop an immunohistochemistry (IHC) assay for ASNS. As with the NanoPro assay, optimized IHC conditions yielded a large dynamic range of staining intensity, and staining was completely ablated by ASNS siRNA. To test the hypothesis that subsets of various cancer types express very low levels of ASNS, we have initiated ASNS IHC of more than 20 tissue arrays representing a wide variety of cancer types. Using a 3-point scoring system (0 = negative, 1 = low, 2 = high), among the tumor samples assayed, 90/136 (66%) of bladder cancer, 63/133 (47%) of bone cancer, 32/149 (22%) of breast cancer, 29/115 (25%) of brain cancer, 51/168 (30%) of colon cancer, 2/85 (2%) of endocrine system cancer, 23/99 (23%) of liver cancer, 7/64 (11%) of head and neck cancer, 7/136 (5%) of lung cancer, 13/53 (25%) of lymphoma, 1/25 (4%) of bone marrow lymphoma, 2/35 (6%) of lymphoma from spleen, 9/109 (8%) of melanoma, 81/396 (21%) of ovarian cancer, 3/29 (10%) of uterine cancer, 27/73 (37%) of pancreatic cancer, 5/119 (4%) of prostate cancer, 10/125 (8%) of renal cancer, 25/138 (18%) of testicular cancer, and 8/39 (21%) of thyroid cancer were ASNS-negative (score = 0), suggesting that a subset of each cancer type may be sensitive to the drug L-asparaginase. Efforts are underway to apply the NanoPro assay to the NCI-60 cell line panel and to continue performing ASNS IHC to survey tissue arrays for ASNS expression. |
 |  | The use of nanoimmunoassay (NIA) technology to predict response to
insulin‐like growth factor‐1 receptor (IGF1R) inhibition in head and neck squamous cell carcinoma (HNSCC) (University of Virginia, AACR 2012) | [show details] |
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Background: Signaling from the IGF1R plays a role in resistance to anti‐cancer therapy in HNSCC. Thus, targeted inhibition of the IGF1R holds substantial therapeutic potential. While several inhibitors of the IGF1R are in clinical trials, there is no biomarker that predicts tumor responsiveness to anti-IGF1R therapy. Such a predictive biomarker is likely to be a component of the most prominent downstream signaling cascades from the IGF1R, which include the MEK/ERK or PI3K/AKT pathways that principally regulate proliferation and survival, respectively.
Hypothesis: Short‐term changes in the activation status of downstream signaling proteins will be predictive of long‐term tumor response to inhibitors of the IGF1R, and these changes will be detectable in minimal tissue samples using NIA technology. |
 |  | Use of Nano-ImmunoAssay to Generate Rapid, Quantitative Nanoscale Proteomic Profiling of the Hypoxia Pathway in Renal Cell Carcinoma Clinical Specimens (Stanford University, ASCO 2012) | [show details] |
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Novel inhibitors of the hypoxia pathway [VEGF, PDGF] achieve response rates of 30-57% in renal cell carcinoma (RCC); yet threshold levels of targets and downstream signaling proteins have not been identified as biomarkers to guide treatment.
Methods: To profile hypoxia proteins in RCC clinical specimens, we have developed the use of automated nanoscale immunoassays for charge-based protein separation (NIA, NanoPro 1000) and charge-based
protein separation (Simple Western, Sally). To decrease the amount of tissue and invasive procedures required to obtain cells for analysis, we optimized assays to profile specimens acquired by fine needle aspiration (FNA).
Results: We used Simple Western to quantify proteins of the MAPK (ERK1, ERK2, pERK1, pERK2, MEK2), PI3K (S6, GSK3b, AKT2, pan-AKT) and STAT pathways (p-STAT5) and loading controls (tubulin, HSP-70) in more than 200 FNA's from solid tumors including RCC. Profiles can be completed overnight after receiving the specimen. Unique to NIA, we also analyzed percent phosphorylation and resolved differences in even a single phosphorylation in FNA specimens, allowing us to group tumors based upon different patterns of phosphorylation and percent phosphorylation.
Conclusions: Rapid and quantitative nanoproteomic profiling in very small amounts of clinical specimen is enabling translational studies for novel diagnostic and predictive biomarkers. |
 |  | Size and Charge Based Analysis of ERK1/2 and 4E-BP1 Using Automated Capillary-Based Systems for Nanoscale Protein Analysis | [show details] |
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Aberrant expression and signaling in the EGF signaling cascade is a common occurrence in a variety of cancers including breast cancer. Understanding how EGF signaling impacts disease progression is key to the development of novel therapeutics. Analysis of ERK1/2 and 4E-BP1 expression in cancer samples frequently employs Western blot analysis. In-depth phosphorylation analysis often requires 2D gels which are extremely variable and labor intensive, followed by MS analysis. In this study, novel, capillary-based technologies by ProteinSimple are used to evaluate changes in signaling proteins. Size-based as well as charge-based separation techniques were utilized, each of which is followed by immunoassay detection. |
 |  | A Fully Automated Capillary Electrophoresis System for Western Analysis Using On-Line Stacking as an Easy Tool for the Enhancement of Sensitivity and Resolution | [show details] |
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Size-based characterization of proteins has been predominately performed by either SDS-PAGE/Western blot analysis or by capillary electrophoresis (CE). Each technique has advantages Western blotting exploits high sensitivity as well as specificity of antibody binding, and CE offers high resolution and reproducibility. The sensitivity and resolution of the results obtained from either method is often challenged by the ability to preconcentrate or stack enough protein sample before separation. Simon, a new size-based separation platform that runs Simple Western assays, combines for the first time the advantages of both Western blotting and CE into a single automated workflow. The work presented illustrates the dependency of stacking efficiency upon the plug length of the sample and stacking matrix. Optimization of stacking conditions resulted in a significant sensitivity and resolution increase using the Simple Western assay. |
 |  | Comparison of Conventional Western Blot Analysis with a Fully Automated Capillary Electrophoresis System for Size-based Analysis of the AKT Pathway Signaling Cascade | [show details] |
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We present data comparing analysis of key targets of the AKT signaling cascade via Western blot and Simple Western on Simon, highlighting workflow, biological response, sensitivity, and resolution. |
 |  | Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161. Novartis Poster AACR 2011. | [show details] |
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Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161 |
 |  | Assay Development on the NanoPro Platform: 4E-BP1 and 4E-BP2 (SBS 2010) | [show details] |
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We present the development of novel nanoimmunoassays for the translational repressor proteins 4E-BP1 and 4E-BP2 using NanoPro technology. Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase pathway regulate 4E-BP1 activity, making 4E-BP1 a focal point for these two important signaling pathways. 4E-BP2 regulation is poorly understood, partially due to the lack of specific anti-phospho 4E-BP2 antibodies. Our assay, developed on the Cell Biosciences NanoPro platform, enables detailed differential investigation of 4E-BP1 and 4E-BP2 phosphorylation and signal transduction. |
 |  | A rapid screening method for monitoring signaling changes in the monocyte cell line U937 (AACR 2009) | [show details] |
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Here we describe a precise screening assay that quantifies changes in phosphorylation of proteins in samples from as few as 100 cells, which is simple, rapid, and relatively low in cost. A nano-immunoassay system (Cell Biosciences) was used to measure changes in expression and activation of relevant signaling proteins, including MEK, ERK and STATs in U937 monocyte cells before and after cytokine treatment. A single pan-specific antibody was used to distinguish between the phosphorylated and non-phosphorylated protein isoforms, as the nano-immunoassay (NIA) method separates different phosphorylated forms of a protein based on their isoelectric point. In parallel, phospho-protein FACS analysis, which is the current state of the art for measuring multiple signaling pathways, was performed to compare changes in expression and phosphorylation of the signaling proteins. Phospho-protein FACS analysis is expensive and requires considerable technical expertise, which limits its application for large numbers of samples. This novel nano-immunoassay screening method is currently being employed at the Stanford Human Immune Monitoring Core (HIMC) and is being used for high-throughput screening of compounds that influence monocyte activation, monocyte/macrophage differentiation and analysis of various disease states in small primary tissue samples. Practical examples will be given. |
 |  | Application of a nanoimmunoassay platform to assess changes in EGFR-dependent signaling pathways in lung cancer cell line: surgical resections and laser-capture microdissection from patient-derived tumor cells exposed to EGFR tyrosine kinase inhibitors (AACR 2009) | [show details] |
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To fully enable the vision of 'bench-to-bedside' requires the development of not only novel therapies, but novel techniques for evaluating their efficacy in cell lines, animal models and primary tumor material. This report describes our development of a technique employing a nanoimmunoassay platform for the analysis of signaling protein activation in primary non-small lunch cancer (NSCLC) solid tumors. |
 |  | Adoption Success: Expansion Of Rapid Screening for Monitoring Signaling Changes (AACR 2009) | [show details] |
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Cell Biosciences' novel nano-immunoassay screening method is being adopted in an ever-increasing range of institutions, thanks to an innovative collaborative effort collaborative effort between Stanford's Human Immune Monitoring Core (HIMC), Comprehensive Cancer Center, and Cell Biosciences.
The precise screening assay quantifies changes in phosphorylated and non-phosphorylated protein isoforms in tiny samples. Notably, the assays are simple, rapid, and relatively low in cost.
The centralized location of the instrument, and unique collaborative environment have enabled rapid development and adoption of Firefly assays - first within Stanford, and now extending to other institutions, both academic and commercial. |
 |  | Independent Measurement of MEK Phosphoforms by Capillary Immunoassay (AACR 2008) | [show details] |
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Activation of the MAPK pathway involves a complicated web of MEK phosphorylations. The two MEK isoforms are regulated by at least 3 other enzymes — PAK, RAF and ERK. Up until now it has been impossible to quantitate and determine the stoichiometry of the various multiply phosphorylated MEK forms. We have developed a new capillary immunoassay which resolves the different MEK variants, and allows measurement of the relative abundance of each form with a single antibody. This measurement of how the multiply phosphorylated forms change gives insight into how the dynamics of pathway feedback and activity change in response to drug treatment.
We have shown that signaling upstream of MEK kinase is inhibited by negative feedback in tumor cells in which the pathway is driven by HER kinases. In these cells, MEK1 is phosphorylated at ERK-- and PAK-dependent sites (T292, S298), whereas phosphorylation on RAF-dependent sites is undetectable. A selective MEK inhibitor inhibits ERK phosphorylation, relieves the negative feedback and activates MEK phosphorylation in these cells. Under these conditions, phosphorylation of both kinases on the RAF dependent sites (S217, S222) is markedly induced. Thus, inhibition of MEK/MAPK signaling in these cells abrogates upstream feedback of the pathway and results in a complex change in phosphorylation of MEK due to multiple kinases.
The capillary immunoassay allows determination of complex changes in phosphorylation of MEK kinase by PAK, RAF and ERK kinases in response to MEK inhibition. This technique will be useful in mapping pathway network response to targeted drugs in vitro and in vivo. |
 |  | Measurement of Oncoproteins in Primary Hematopoietic Malignancies Pre- and Post-Therapy Using a Nano-Immunoassay System (AACR 2008) | [show details] |
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Oncoprotein quantification in clinical specimens is important for the diagnosis of specific hematopoietic malignancies as well as the development and monitoring of effective therapies that target oncoproteins. Current protein detection methods require large samples, precluding routine serial tumor sampling to assess changes in oncoprotein levels. Here we demonstrate the use of a nano-immunoassay system (Firefly™ system) to distinguish between patient specimens of Burkitt's vs. Follicular lymphoma by characterizing patterns of MYC and BCL2 expression. Changes in the expression and activation of a variety of onco/signaling proteins including ERK, MEK, STAT and JNK in malignant hematopoietic (CML) cells and patient samples before and after treatment with therapeutic agents that impact oncogenic signaling pathways are also shown. The expression levels of the different proteins were measured with high sensitivity in samples as small as 400 cells. A key benefit of this technology is that it separates protein isoforms based on its isoelectric point. Using this assay, we were able to distinguish and quantify the phosphorylated and non-phosphorylated forms of each protein with a single antibody. Thus, we have developed a novel technique which can precisely evaluate the activity levels of signaling proteins in oncogenic pathways from very small samples. |
 |  | Measuring Tyrosine Kinase Inhibitor Effects on Cell Signaling Pathways (Beatson 2008) | [show details] |
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A recent wave of anti-cancer compounds that target tyrosine kinases (TKIs) has been moving through the drug development pipeline. Assessment and screening of lead compounds in simple model systems is relatively straight forward. Until recently, however, determining the impact of these compounds in complex biology of patient-derived cells and tissues has been diffcult. Proposed genetic or protein biomarkers can act as surrogates to a response, but measuring the signaling pathway in both the target cells and surrounding normal tissue will provide a more direct metric. This has proven diffcult due to the limited nature of primary material and complexity of tissue structure. Here we describe a novel nano-immunoassay platform (Firefly™) that has two significant advantages over traditional immunoassays: (1) extremely sensitive protein detection, and (2) physical isoform separation, which allows for quantitation of protein isoforms as well as post-translational modifications such as phosphorylation.
Applications of this technology that will be described include:
1. Effect of TKIs on signaling in punch biopsies of non-small cell lung cancer cells
2. Signaling pathway response from chronic myleogenous leukemia patients to therapies targeted to the bcr/abl translocation |
 |  | Protein Expression and Cell Signaling Quantified in Rare Cells (ISSCR 2008) | [show details] |
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The discovery of tumor stem cells in acute myeloid leukemia a decade ago initiated a field of research has accelerated growth in the past few years. Researchers are now describing tumor stem cells in a variety of hematopoietic and solid tumors. The impetus for much of this research is the desire to identify targets for drug intervention in these critical tumor populations. The molecular pathways that functionally define these cells are important therapeutic targets. Tumor stem cells are rare and provide insufficient material to use standard assay methods. Although DNA microarray and/or qPCR are used to study tumor stem cells, their rare nature limits quantitative protein analysis. This creates a gap in our knowledge since many proteins, such as beta-catenin or MAPK signaling proteins, are not regulated at the transcriptional level, but through post-translational modifications (phosphorylation, ubiquitination, etc.). Here we describe a technique utilizing a nanoimmunoassay platform (Firefly?) to measure tumor stem cell proteins. Transitional tumor stem cells (TCC+) were sorted from a patient tumor and lysed for analysis. A lysate of 400 cells was subjected to isoelectric focusing and immobilization. Immunodetection was performed and quantitation of signal was measured using HRP-labeled secondary chemiluminescence reagents. Here we report beta-catenin protein concentrations of 192 ng/mg of total protein in the tumor stem cells, which was undetectable in 'non-stem' tumor cells. Comparisons of protein levels and the degree of phosphorylation are made between these samples, other tumor cell lines and hematopoetic stem cells. |