Simple Western technical library

Industry-leading fluorescence sensitivity for Western blot analysis

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).

Comprehensive monitoring of VP1, VP2, and VP3 protein concentrations and their molar ratios is an effective tool to ensure initial optimization and consistent quality of the final AAV gene therapy products. Here we introduce PROGEN´s recombinant VP standards used with Simple Western™ automated capillary electrophoresis in fit-for-purpose solutions for VP capsid protein ratio and viral protein titer measurements. We further show the establishment of the AAV2 VP protein standards by PROGEN as well as the assay performance data that demonstrates linearity and high sensitivity in an automated workflow optimized for AAV samples.

Industry-leading fluorescence sensitivity for Western blot analysis

Simple Western and Single-Cell Western have the potential to make significant contributions to three important branches of immuno-oncology: angiogenesis of the tumor microenvironment, immune checkpoint inhibition, and chimeric antigen T-cell (CAR T-cell) therapy. Learn more.

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.

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.

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.

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.

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.

Meet Abby™, the next generation chemiluminescence western protein analyzer who moves your Western workflow into the 21st
century. Abby 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 Abby does the rest. She separates your proteins by size and precisely manages antibody additions, incubations, washes, detection steps, and even the analysis. Come back to fully analyzed and quantitated results of 24 samples in as little as 3 hours. With Abby’s chemiluminescent detection you’ll get
picogram-level sensitivity, letting you maximize the data you get from your sample. Best of all, you get reliable and reproducible
results with target normalization to the amount of protein loaded.

Analysis is a breeze. Want to identify whether a protein is present or absent? Abby gives you the qualitative Western data you are
used to seeing. Even better, she’ll quantitate the results for you too. With just a few clicks you’ll be analyzing immunoassay-like
standard curves and precisely quantifying your protein. Annotate your data directly in Compass for publication ready images. Abby,
she’s the next generation Western super-hero.

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.

This eBook provides protocols for the analysis of more than 30 proteins by Simple Western Charge, including common components of signaling pathways, enzymes, membrane proteins, and more.

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 automated protein analysis with Simple Western can be performed to study cell signaling for regenerative medicine, resulting in high-quality and reproducible data.

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.

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.

ProteinSimple supports Parkinson’s disease research. See how Wes does Simple Western protein analysis on Parkinson's disease samples.

“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.”

“[Wes] Provides quantitative data, saves time and uses far less sample.”

"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."

“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

"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

“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

"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

Checklist: Abby Site Preparation

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.

Purity is a critical quality attribute (CQA) that must be monitored during AAV manufacturing. Impurities in protein products can be dangerous and impact efficacy. For example, protein impurities in final drug products may lead to undesirable immune responses in patients, so detecting total protein is critical for revealing impurities in preparative protein production. Traditional methods for total protein detection rely on SDS-PAGE with dyes like Coomassie Blue, or more sensitive stains like SYPRO Ruby and silver stain. However, SDS-PAGE requires large sample volumes, a lot of hands-on time, and it is poorly reproducible. Also, the use of staining dyes often comes with a lot of waste and can require specialized imaging equipment to which not every researcher has access.

Here, we present a new workflow that enables total protein detection of AAVs on the Simple Western capillary electrophoresis-based immunoassay platform with a sensitivity that exceeds SYPRO Ruby. While SYPRO Ruby requires at least 1 ng of protein for reliable detection, Simple Western can reliably detect as little as 150 pg. These findings should enable researchers who are currently using SDS-PAGE to monitor purity to apply the automated platform and sensitivity improvements enabled by this workflow to assess AAV purity using smaller AAV sample sizes.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Product Insert: Abby Training Kit

This Serology Module provides all the reagents needed to detect SARS-CoV-2 IgG antibodies in human serum or plasma in a Simple Western size-based assay when paired with a Separation Module. Please note that this module can only be used with Jess and Wes systems and is for research use only.

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1次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。

Jess Protein Normalization for Immunoassay Module

These Detection Modules provide all the reagents needed to detect your target of interest with a primary antibody in a Simple Western size-based assay when paired with a Separation Module.

These Detection Modules provide all the reagents needed to detect your target of interest with a primary antibody in a Simple Western size-based assay when paired with a Separation Module. Please note these Detection Modules can only be used with Jess.

See why leaders in regenerative medicine and cell therapy research use Simple Western™ and Single-Cell Western.

This Scientific Review highlights how Simple Western and Single-Cell Western are instrumental in developing cell & gene therapies for Duchenne muscular dystrophy, lupus, cystic fibrosis, and sickle-cell disease. We also include references to other noteworthy cell & gene therapy publications that harness the power of Simple Western and Single-Cell Western.

Simple Western provides new vaccine technology to combat outbreaks. Simple Western instruments outperform traditional Western blot at every stage of the vaccine development pipeline.

To characterize the efficacy of Degrader molecules, researchers generally run dose response curves by way of traditional SDS-PAGE Western blotting methods. Simple Westerns let you separate and analyze proteins by size (or charge) from 2 kDa to 440 kDa in just 3 hours.

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

Since its development in 1979, 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.

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

We enable cutting-edge research to uncover the role of proteins in cancer and provide novel approaches to develop and
analyze protein-based therapeutics. We empower you to make your next discovery by eliminating common protein analysis
workflow challenges.

ブロットカートリッジを用いてJessでブロットイメージング
クイックリファレンスガイド

Blot imaging on Jess using the blot cartridge Quick Reference Guide

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.

Learn how Simple Western can characterize full signaling pathways with little sample volumes, offering picogram-level sensitivity, and high-throughput analysis overnight.

ＨＤＲはHigh Dynamic Range(高いダイナミックレンジ)を表します。写真撮影においては最初に露出を変えながら複数の画像を撮影した後で、特殊なアルゴリズムを用いて、これらの画像のベスト部分を組み合わせて自動的に最良のイメージを作成します。 SimpleWestern®のＨＤＲも同様なアルゴリズムに基づいています。 ダイナミックレンジが広がると、貴重なサンプルを無駄にしたり、アッセイの最適化に多くの時間を取られることもなくなります。アッセイの最適化の時間を短縮するために、アッセイの検出ステップとデータ解析の両方を強化するＨＤＲ検出プロファイルを利用してWesのダイナミックレンジを拡大しました。

Jess, the newest Simple Western™ technology from ProteinSimple, allows you to not only run fully automated capillary-based Westerns, but she takes digital images of traditional chemiluminescent Western blot membranes. ProteinSimple’s AlphaView® software allows you to analyze Western blot images taken on Jess so that you can prepare publication-ready figures. In this tech note, we’ll show you how to export your images from Jess into AlphaView, and we’ll cover the tools that you’ll need to get the most out of your Westerns.

If you’re experiencing primary antibody derived cross reactivity with the 230 kDa protein used as one of fluorescent standards, we’ve got a solution.

In this technical note we show you how this new assays works and give you a couple examples comparing Simple Western performance with traditional Western.

Multiplexing two targets in one capillary with Simple Western doubles the number of data points per sample and increases the data quantitation accuracy when you normalize to biological loading control or system control.

When you're ready to start multiplexing, first optimize the primary antibodies separately to find the saturating dilution for each. Then combine them in Antibody Diluent 2 to detect the two targets in the same capillary. If the primary antibodies are raised in two different host species, you'll also need to combine two different anti-species secondary antibodies. But mixing Ready-To-Use (RTU) secondary antibodies will dilute them and cause the signal to decrease or become more variable because they're no longer saturating. We've got the situation under control with the 20X Anti-Rabbit HRP Conjugate.

An immunoassay with a large dynamic range lets you simultaneously see really high and really low signal at the same time. This means not needing to spend time either diluting or concentrating your samples to get your protein of interest in the detected dynamic range. Wes® has always been great at detecting really low levels of protein and now we’ve improved what you’ll see on the high end, giving you at least one more log in dynamic range compared to a Traditional Western blot.

User Guide: Abby

This user guide will provide you with information on how Simple Western assays work as well as other useful operating and installation information.

Here we show two case studies illustrating the Bio-Techne dTAG workflow. We present data to demonstrate the value of testing both CRBN- and VHL-recruiting dTAG Degraders for each POI target as well as the use of negative control Degraders. We also show the utility of using a FKBP12 antibody for detecting POI∙dTAG fusion molecules, allowing for a more simplified
procedure when probing multiple targets.