Simple Western technical library

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.

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).

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.

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).

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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.

ProteinSimple offers a range of research tools to study the biological processes that accompany neurological/neurodegenerative disease.

Meet Jess, your protein analysis problem solver. Jess automates the protein separation and immunodetection of traditional Western blotting, eliminating many of the tedious, error-prone steps. Just load your samples and reagents into the microplate and Jess does the rest.

Protein analysis comes with many challenges—labor-intensive protocols increase time to result and multiple hands-on steps increase user error and data variability. At best, you end up with Western blot-like,
semi-quantitative results when what you really need, and deserve, is highly reproducible immunoassay quantitation. Discover more and solve your protein analysis problems with Simple Western.

Electrophoresis eBook

In this eBook, we examine technological advances in protein analysis that are at the forefront of immuno-oncology research and therapeutic development.

Research into prevention and treatment of disorders of the brain and nervous system are in greater demand as the world’s population expands. This group of diseases result in more hospitalizations than any other group, including heart disease and cancer. Odds are that you know someone affected by a neurological disorder, as The World Health Organization estimates that one billion people are affected by them, contributing to 11% of the world’s disease burden.

Proteins are the heart and soul of functional biology and understanding proteins is central to understanding disease. However, proteins are difficult to interrogate because they are large, complex, and unique. Here at ProteinSimple, we believe that traditional protein analysis tools can be overly complex or inadequate, and our goal is to make protein analysis simpler, more quantitative, and affordable. Ultimately, we want to help researchers gain a better understanding of proteins and their role in disease.

Learn how Jess furthers therapeutic research of human gliomas. Reduce preparation time while saving reagents, samples, and antibodies with target optimization.

Aurelia Bioscience is solving drug discovery challenges by taking on PROTAC studies with ProteinSimple’s Jess and Simple Western assays.

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.

“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.”

"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."

“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 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

"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

"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

”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

We want to keep you updated on all of our advances in protein discovery and analysis, so here is our quarterly newsletter. It's never been easier to see what we're up to because each page covers a product platform and, if you want to attend an event, all you have to do is click on it to register. From new application notes and publications to product updates, we've got you covered.

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.

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.

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).

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.

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.

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.

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.

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.

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-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.

Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161

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.

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.

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.

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.

これらのDetection Moduleは、Separation Moduleと一緒に使用し、分子 量に基づいたシンプルウェスタンで、抗体を用いてターゲット分子を検 出するのに必要なすべての試薬を含んでいます。

1. Separation Module preparation
2. Detection Module preparation
3. Pipette your plate
4. Start Jess or Wes

• List of Related Consumables

Product Insert: Jess Training Kit

Simple Western Analysis on Jess Reveals a Link Between Elovanoids and ACE2 Expression for COVID-19 Research

Since its development in 19791, the
essential steps of the Western blot (gel electrophoresis, transfer and immunodetection) have remained constant with only minor modifications. With the introduction capillary based westerns in 2009, ProteinSimple has been the world leader in revolutionizing traditional western blotting through
advances in automation, sensitivity, and single-cell resolution among others.

The power of Simple Western platforms, like Peggy Sue™, Sally Sue™, and NanoPro 1000™, has been harnessed in many fields of research including cancer, drug discovery, stem cells and regeneration, and cell and gene therapy. Below are just a few examples of how researchers are using Simple Western assays to make important scientific breakthroughs.

Simple Western Charge assays on Peggy Sue™ or NanoPro 1000™ analyze up to 96 native, non-denatured samples in a fully automated fashion, and the data generated are reproducible and quantitative. Simple Western Charge assays use as little as 0.2 μg/μL of protein in just 5 μL of sample, and those few microliters can be interrogated up to eight times! This means you’ll get a large amount of data from a very small sample size.

シンプルウェスタンTotal Protein Assayを用いたCompassソフトウェア
クイックリファレンスガイド

SDS for Protein Normalization Assay Module for Jess

A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.

In this technical note, we’ll walk you through fluorescent multiplexing on Jess and provide you with guidance for developing an assay efficiently.

Block non-specific signal and prevent cross-reactivity when using anti-goat secondary antibodies with our Anti-Goat Detection Module for Wes, Peggy Sue and Sally Sue

There is a good reason why it is called apparent molecular weight. SDS-PAGE and Simple Western separate proteins based on the amount of SDS bound to the protein during denaturation. The correlation between motility and molecular weight (MW) is based on the uniform binding of negatively charged SDS to the protein (1.4g SDS/g Protein) which results in a constant charge-to-mass ratio. In reality, the amount of SDS bound is anything but constant and it’s affected by many factors...

We know that your samples are usually the most precious part of an experiment. It can take a lot of time and resources that add to the expenses needed to grow and treat cells or months to raise and treat mice. But when researchers sit down to calculate the cost per data point they often forget to factor in the sample and focus more on things like buffers, gels, membranes, and antibodies. Plus, nobody wants to use up the limited amount of sample they have in one experiment just to have to take the time to make more samples when it's all about publishing or perishing.

Simple Western®, an immunoassay or total protein assay performed in a small capillary, has been the perfect solution when it comes to saving samples. And now we've just increased your sample savings by decreasing the amount of sample you need for each experiment.

HDR stands for high dynamic range and in photography it works by first taking a series of images with different exposures. Some cool math automatically combines the best parts of these images to create the best picture. HDR on Simple Western isn't all that different. And with a large dynamic range, you won't have to waste precious samples running assay optimization experiments. To reduce assay optimization time, we've expanded the dynamic range for Wes with a high dynamic range detection profile that enhances both the detection step in the assay and the data analysis.