Simple Western technical library

The epidermal growth factor receptor (EGFR) is a transmembrane receptor for the epidermal growth factor family (EGF-family) of extracellular protein ligands. Ligand binding activates its intracellular tyrosine kinase domain resulting in auto as well as substrate phosphorylation. Mutation and deregulation of EGFR is implicated in many cancer types. The data shows a time dependent change in anti-phospho EGFR (Y1068) antibody signal in HeLa cells in response to EGF treatment.

The p21 activated kinases (PAK) proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1, and have been implicated in a wide range of biological activities. PAK2 is a member of the PAK subfamily 1 including PAK1 (α-PAK), PAK2 (γ-PAK, PAKθ, hPAK65), and PAK3 (β-PAK). The kinase domains within a subfamily show a high degree of sequence identity, and all PAK proteins bind GTP-bound Rho family members at the amino-terminal p21-binding domain (PBD). Our data in HeLa cells show a pattern of 5 peaks using a pan PAK1/2/3 antibody of which two are picked up consistently by 3 different specific PAK2 antibodies identifying them as PAK2 peaks.

GSK3 is a critical downstream element of the PI3 kinase/AKT cell survival pathway whose activity can be inhibited by AKT-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β. While GSK-3α and GSK-3β have high sequence homology, their biological function differs. The data presented shows an increase of peaks detected by anti-phospho GSK-3α antibody as well as an increase of the same peaks detected by the total anti-GSK-3α antibody in response to EGF treatment in MCF10A cells. At the same time, the peaks not recognized by the anti-phospho Ser21 antibody decrease in size, implying that these peaks represent non-phospho or non-pS21 phospho GSK-3α forms. The position of these peaks around pI 9 is in accordance with the theoretical pI for this sequence. In other cell systems (A549 for example), both the anti-total GSK-3 antibody as well as the anti-phospho GSK-3α antibody also recognize peaks around 6.0. Alignment of the GSKα and GSKβ profiles in addition to use of antibodies recognizing both isoforms confirmed the specificity of the anti-GSK-3α versus the anti-GSK-3β antibodies used (data not shown).

Thioredoxin acts as an antioxidant and is found in nearly all known organisms. It exists in two isoforms and presents a double peak around pI 4.6. The data presented show the utility of Thioredoxin as a loading control for EGF treated A549 and HeLa cells.

The 70 kilodalton heat shock proteins (Hsp70) are a family of ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms. The Hsp70s are an important part of the cell's machinery, and help to protect proteins from misfolding under stress. We describe its use as loading control for EGF stimulation in HeLa and MCF10A cells. In the NanoPro assay, Hsp-70 is present as a single peak around pI 5.8 under the conditions described.

β-2-Microglobulin, also known as B2M, is a component of MHC class I molecules, which are present on all nucleated cells. In the NanoPro assay, it presents a single peak around pI 6. The data show its application as a loading control for EGF stimulation in HeLa cells as well as MCF10A cells.

The Signal Transducer and Activator of Transcription (STAT) proteins regulate many aspects of cell growth, survival and differentiation. The transcription factors in this family are activated by Janus Kinase (JAK). Dysregulation of this pathway is frequently observed in primary tumors and leads to increased angiogenesis, enhanced survival of tumors and immunosuppression. STAT5 is constitutively active in the overexpressing BCR-ABL K562 myelogenous leukaemia cell line. Imatinib (also known as Gleevec®), a BCR-ABL inhibitor, reduces STAT5 phosphorylation in these cells. STAT5 is also part of the Epidermal Growth Factor (EGF) signaling cascade as shown in MCF10A cells.

Extracellular signal-regulated kinases (ERK) or classical MAP kinases are widely expressed intracellular signaling molecules involved in regulation of meiosis, mitosis, and post-mitotic functions in differentiated cells. Many different stimuli (including growth factors, cytokines, virus infection, ligands for heterotrimeric G protein-coupled receptors, transforming agents, and carcinogens) activate the ERK pathway. We show an example of ERK phosphorylation in MCF10A cells in response to treatment with epidermal growth factor (EGF).

Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and intracellular signals. While the functions of MEK1 and MEK2 are very similar, theses kinases differ significantly in the way they are regulated. For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK2 activation in MCF10A cells in response to EGF stimulation.

c-Jun N-terminal kinases (JNK), originally identified as kinases that bind and phosphorylate c-Jun on Ser63 and Ser73, are mitogen-activated protein kinases which are responsive to stress stimuli, such as cytokines, UV-irradiation, heat shock, and osmotic shock, and are involved in T cell differentiation and apoptosis. JNK1, 2 and 3 share a total of 10 isoforms with pI's ranging from 5.4-7.6. All three JNK kinases share a similar Thr/Tyr phosphorylation site (T183/Y185). We evaluate change in JNK phosphorylation in UV-treated HEK293 cells and imatinib-treated K562 cells using a dual phospho-antibody against that site.

Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been shown to potentially play a role in energy homeostasis. We show 4E-BP1 activation in MCF10A cells in response to EGF using total and anti-phospho 4E-BP1 antibodies that enable distinction between phospho and non-phospho peaks.

Crk-like protein (Crk-L) is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. It has been shown to activate the RAS and JUN kinase signaling pathways and transform fibroblasts in a RAS-dependent fashion. Crk-L is a substrate of the BCR-ABL tyrosine kinase and plays a role in fibroblast transformation by BCR-ABL. We show that Crk-L phosphorylation is reduced in response to Imatinib (commonly known as Gleevec®) treatment in K562 cells.

Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The FLAG-tag
consists of a short peptide sequence (MADYKDDDDKM). EGFP was expressed with a FLAG-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.

The enzyme-drug, L-asparaginase, has been used since the 1970s to treat acute lymphoblastic leukemia. Asparagine synthetase (ASNS) expression has been found to be correlated with L-asparaginase efficacy in leukemia cell lines, in leukemia primary tumor samples, and more recently, in cancer cell lines from other tissues of origin. Silencing ASNS expression by RNAi has indicated the L-asparaginase/ASNS relationship is causal and suggests that ASNS expression may be useful as a predictive clinical biomarker of L-asparaginase efficacy. ASNS presents as a single peak in the NanoPro assay. Expected changes of expression are observed upon siRNA as well as L-asparaginase
treatment.

cIAP1 is a member of the inhibitor of apoptosis (IAPs) family of proteins. It is up-regulated in several human cancers and plays an important role in tumor survival. cIAP1 functions to prevent cellular apoptosis by preventing the activation and/or inhibiting the function of different caspases.

Eukaryotic elongation factor-2 (eEF2) catalyzes ribosome translocation during translation of mRNA. Phosphorylation of eEF2 by eEF2 kinase (also known as CaM kinase III) inactivates the protein and can block protein synthesis. Stimulation of growth is associated with a decrease in eEF2 phosphorylation. Inhibition of eEF2 phosphorylation in the hippocampus has recently been associated with an anti-depressant eect. We show that insulin treatment reduces phosphorylation of eEF2 in H4IIE cells. Inhibition of phosphorylation of eEF2 is restored in the presence of Nh125.

Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) are two highly homologous mitochondrial outer membrane proteins necessary for mitochondrial fusion. Mitochondrial fusion is necessary to maintain mitochondrial health and function, and mitochondrial dysfunction is implicated in diseases such as Charcot-Marie-Tooth 2A and dominant optic atrophy, as well as in multiple tissue types. Phosphorylation of these proteins has not been widely studied. Our data show detectoin of Mfn2 protein in three neuronal tissues: retina, optic nerve, and the superior colliculus region of the brain in a mouse model, DBA/2J, a widely accepted model for glaucoma.

The Mitogen Activated Protein Kinase p38 is activated by stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation and apoptosis. We show that Interleukin-1 (IL-1) treatment induces phosphorylation of p38 in fibroblasts. In addition, two independent antibodies recognize peaks at pIs 5.4 and 5.8 for phosphorylated p38.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor protein that is frequently mutated or deleted in many tumors. PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PtdInsP3) inhibiting phosphoinositide 3-kinase (PI3K) activation of AKT repressing cell growth, proliferation and survival. Recent studies show that PTEN phosphorylation alters its stability and function.

Acetylation is a common post-translation modification typically observed on chromatin proteins as well as other regulatory proteins and metabolic enzymes. The reaction is catalyzed by N-terminal acetyltransferases, occurs predominantly during protein synthesis and appears to be irreversible. Pan-Ac-Lysine antibodies are widely used for detection of acetylation of imunoprecipitated proteins when no specific antibodies are available.

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.

In this application note, we simultaneously assess p-Akt and total Akt levels using different detection channels combined with in-capillary protein normalization to demonstrate the superplexing power Jess possesses.

シンプルウェスタンを用いてハイスループットで 糖鎖のキャラクタリゼーション

In this application note, we focus on the accurate detection of four candidate contaminants that may be present during various stages of the therapeutic protein and vaccine development processes: host cell protein (HCP), Protein A, green fluorescence protein (GFP) and bovine serum albumin (BSA).

In this how-to-guide, we'll show you how to use the Total Protein Detection Module (DM-TP01) with any immunoassay detection module of your choosing to get total protein and immunoassay data in the same run.

Laser capture microdissection (LCM) is a powerful tool to identify and isolate a pure sample of the cell type you're
interested in. But, proteomic studies with LCM samples are really restricted by the small amounts of tissue collected with
each capture since there just isn't much to work with. You often have to use the entire sample captured for traditional
Western blot analysis and that only leaves you with one data point! So, researchers often use 2D Electrophoresis and
mass spectrometry instead to max out the amount of data they can generate. Both of these methods have their own
limitations when it comes to ease-of-use and reproducibility though. Immunohistochemistry is also used to analyze LCM
samples as it's a more accessible technique, but it really doesn't give you a lot of info either.

Simple Western is an automated capillary-based immunoassay that changes the proteomic research game. You only
need 1-10 µL of LCM sample for each data point, so you'll get more data points for each sample you collect. Not to
mention the sensitivity that comes with it will even let you analyze proteins you couldn't previously do with traditional
Western blot. And it's all wrapped up in a simple workflow that minimizes your hands-on time. Simple Western is a
sensitive, easy-to-use analytical tool that ups the ante on protein analysis with LCM samples.

In this application note, we'll show you two examples of how Simple Western changed what researchers could do with
their precious LCM samples for the better

The Simple Western Immunoassay is the gel-free, blot-free and hands-free solution for researchers looking for a better way to get their Western blot data. The simple fact that you get analyzed data in just three hours with only 30 minutes of hands-on time changes things forever!

Wes takes the benefits of Simple Western assays a step further by simplifying their workflow with pre-filled plates and disposable capillary cartridges. This application note gives an overview of how to transfer a traditional Western blot assay to Wes.

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.

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.

“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

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.

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

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.

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

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.

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