Simple Western technical library

エクソソーム等、細胞外小胞を準備した際の適切な特性評価では、材料に限りがあり、様々な細胞外小胞の分離方法により、細胞外小胞特有の課題があります。細胞外小胞研究に関する最小限の情報（MISEV）ガイドラインは細胞外小胞タンパク質の含有量について、膜貫通タンパク質、細胞質タンパク質、およびコンタミ由来による非細胞外小胞タンパク質で特性評価することを推奨していますが、MISEVに準拠する簡単で堅牢な分析プロトコルがないため、各ラボで多大な労力を費やしています。ここでは、簡単に細胞外小胞を分離する方法と、MISEV推奨タンパク質の全自動タンパク質分離と免疫検出を紹介します。

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