Technical library

Fujifilm in Morrisville, NC conducted a study to evaluate the Maurice system as a potential platform to replace the iCE3 system for icIEF analysis and the SCIEX PA 800 Plus for CE-SDS analysis. The potential benefits of standardizing on the Maurice system included improved efficiencies and reduced training for scientists, reduced capital investment over time, and better data integrity. This spotlight focuses on their study of the iCE3 and Maurice icIEF application.

Fluorescence-based imaging has grown to be a popular choice for Western blot detection and analysis, owing to its ultra-sensitive,
flexible and convenient capabilities. The FluorChem R Imaging System is compatible with far-red and near-infrared (NIR) dyes and is designed to acquire high-quality
images from fluorescently labeled, multicolor Western blots.

Simple Plex assay for the detection of human HGF in serum and plasma (EDTA/Heparin)

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).

このアプリケーションノートでは新発売のTurbo CE-SDSカートリッジをMauriceで用いると、蛍光標識を必要とせず、タンパク質のサイズと純度のCE-SDSアプリケーションを高速ハイスループット（還元型サンプルは5.5分、非還元型サンプルは8分）で行い、タンパク質の直接検出と高品質のデータを迅速に取得することを示します。Turbo CE-SDS アッセイの高い再現性、分離の直線性、幅広いダイナミックレンジ、優れた検出限界 (LOD) も示します。以前から広く用いられているMauriceのCE-SDSアプリケーションに加え、CE-SDSアプリケーションのスループットの課題を解決し、バイオ治療薬研究開発段階から分析開発やQCまで、ステップごとにニーズのあったタンパク質のサイズと純度分析を1台で行えるようになりました。

このアプリケーションノートでは、AAVプロダクトの安定性と同一性をより理解するためにAAVベクターの電荷不均一性を特性評価する方法を実証します。Mauriceの電荷不均一性解析はAAVの安定性のモニタリングに使用できることも示します。また、熱変性したAAVプロダクトに95°Cで5分間ストレスを与え電荷不均一性解析を行ったところ、酸性側のピークが増加したため、酸性側のピークが増加したAAVは有効性や安全性に悪影響を及ぼす可能性を示唆しています。

このアプリケーションノートでは、RePlex（ストリッピング）を用いて、PI3K/Aktシグナル伝達経路などに関わる分子のトータルタンパク質やリン酸化アイソフォームを検出し定量する方法を示します。このシグナル伝達経路は細胞の成長、生存、アポトーシスを調節し、ヒトの癌では頻繁に変化を生じ、化学療法や放射線抵抗性の原因になっています。Simple WesternのRePlexは固定したタンパク質や、そのエピトープの完全性を損なうことなく、1回目のプロービング後に反応した抗体を効率的に除去することを示します。次に、RePlexを用いて、Aktのリン酸化アイソフォームとパン（汎反応性）Aktに対する抗体でマルチプレックスし、2番目のサイクルで総タンパク質を検出後、データをノーマライズします。 最後に、RePlexの利用範囲を広げて、さまざまな種類の組織でその下流ターゲットやタンパク質発現を特性評価します。

このアプリケーションノートでは、遺伝子・細胞治療の商業化において技術革新の最前線にいるCell and Gene Therapy Catapult (CGTC)が、PROGENの提供するAAVカプシドに対する特異性の高い抗体を使って、製品精製時のAAVカプシドタンパク質のモニタリングや特性評価を、Wes™による完全自動Simple Western™アッセイでどのように行っているかを説明します。

この「how to」ガイドでは、Simple Westernで迅速かつ効率的にマルチプレックスアッセイを行う方法を説明し、高発現タンパク質と低発現タンパク質を同時に観察する方法についていくつかのコツを紹介します。

JessのStellar™ NIR/IR蛍光検出は、優れた再現性と4ログのダイナミックレンジとともに、1pg以下も検出できる業界をリードする蛍光感度をもたらします。このアプリケーションノートでは、細胞シグナル伝達経路を例に、トータルとリン酸化タンパク質アイソフォームを高感度マルチカラー・マルチプレックス検出する方法と、同一キャピラリーで同時にStellar総タンパク質ノーマライゼーションを行い、正確なタンパク質定量を行う方法を示します。

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.

In this Application Note, we developed a novel method for quantifying the DNA content of AAV particles with Simple Western™, a next-generation biomolecular analytical tool that seamlessly combines capillary electrophoresis and immunodetection with conventional Western blot antibodies. Here, the Simple Western method automatically separates AAV samples by Size or Charge followed by specific and sensitive detection using anti-DNA and anti-VP1/2/3 antibodies directly in the capillary for quantitative and reproducible measurement of these central AAV components. For each antibody, we identify the range in which the signal intensity is linearly related to the amount of sample loaded, resulting in a rapid and sensitive assay for accurately quantifying the ratio of % full AAVs to total AAVs in a sample (also known as the Content Ratio).

A growing demand for biosimilars has only added to the fierce competition of drug development. On such a front, time is of the essence and there is an urgent need for robust analytical tools for various stages of biosimilar development. This application note discusses the use of a bi-functional analytical tool, Maurice, in the stability study of Actemra® (Tocilizumab). As a popular monoclonal antibody used for treating rheumatoid arthritis, and more recently, in the treatment of COVID-191, Tocilizumab has several biosimilars in the pipeline of various biopharmaceutical companies. In this study, we demonstrate how Maurice accurately detects and quantitates changes in Tocilizumab under various stress conditions, thus making it a valuable and easy-to-use tool for comparability assessments in biosimilar development.

Utilizing Waters’ Open Interface Portal (OIP) for multi-vendor hardware control, ProteinSimple’s Maurice Empower® Control Kit enables seamless control of the Maurice platform with all key functions preserved and with full regulatory compliance, including 21 CFR Part 11 controls for industry-leading security and data integrity.

Viral capsid content can impact gene therapy product efficacy and is therefore considered a Critical Quality Attribute (CQA) that must be properly evaluated during the development and manufacturing of AAVs. Traditional analytical tools such as transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and ion-exchange chromatography (IEX) can be used to characterize capsid content but are complex, labor-intensive, and pose challenges in data reproducibility, throughput, and scalability. In this application note, we show how imaged-capillary isoelectric focusing (icIEF) technology on Maurice can be used to characterize empty, intermediate and full AAV capsids at native and stability screening conditions, providing robust and reproducible data. With this, Maurice provides crucial data to aid in developing the right formulation for AAV therapeutics.

Polystyrene beads are ideal for particle sizing and sensitivity testing because they easily trigger the MFI imaging detector, yielding consistent and precise sizing and concentration results over a large size range. To ensure correct usage, this application note discusses recommended guidelines on how to use bead calibration for sizing and concentration verification on the MFI 5000 series with or without the Bot1 autosampler.

Abby is the latest Simple Western instrument from ProteinSimple. This instrument automates Western blot analysis and delivers picogram-level sensitivity with her chemiluminescence detection.

In this Application Note, we show how to achieve ultrasensitive total protein detection using the 5X biotin labeling reagent with a focus on AAV analysis.

Here, we present data showing the power of using automated Simple Western platforms to screen panels of Degrader and IMiD compounds in order to quantify degradation activity. In this study, we demonstrate the time savings achieved by automating these large screens as well as Simple Western’s ability to accurately quantify DC50 and Dmax values for specific Degraders and IMiDs.

A multi-antigen serology assay for COVID-19 using Simple Western furthers understanding of the COVID-19 disease progression and the efficacy of a vaccine.

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.

このアプリケーションノートでは、Empower®でMauriceをコントロールし、モノクローナル抗体の画像キャピラリー等電点電気泳動（cIEF）とCE-SDSデータの収集と解析を行います。cIEFデータを時間軸、吸光度、ピクセルを伴う3Dスペクトル(グラフ)で表示すると、一つの3D画像でタンパク質が収束する様子をより包括的に理解できます。

Recent advances in vector engineering, delivery and safety have placed viral vector-based therapy at the forefront of gene therapy, with adeno-associated virus (AAV) being one of the most actively investigated. To support the rise of AAV vectors in the clinic, technological solutions that afford robust quality control assays are essential for implementing Good Manufacturing Practice (GMP), meeting regulatory requirements and ensuring the clinical quality, safety and consistency

In this application note, we validate Milo for the analysis of hiPSC-CM cultures. Further, we demonstrate how Single-Cell Western analysis can track phenotypic marker heterogeneity over time and monitor the relative proportion of cell subsets during culture differentiation

In this application note, you’ll see how CGTC has used highly-specific antibodies exclusively manufactured by PROGEN with fully automated Simple Western™ assays on Wes™ to monitor and characterize AAV capsids during product purification.

In this application note, we’ll show you how Maurice data compares against SCIEX PA 800 systems for reduced and non-reduced CE-SDS separation of a reference monoclonal antibody from the National Institute of Standards and Technology (NIST).

In this application note, we demonstrate how Milo can be used to identify and quantify neural subtypes in a heterogeneous neural sample, and monitor the differentiation of induced pluripotent stem cells (iPSCs) into neurons, astrocytes, and oligodendrocytes using R&D Systems research-grade or GMP differentiation reagents.

MFI Silane-Coated Flow Cell Cleaning and Care Technical Note

このアプリケーションノートでは、バイオ医薬品およびワクチン開発プロセスの様々な段階で存在し得る4つの主要なコンタミ物質(ホストセル蛋白質(HCP)、Protein A、GFP(緑色蛍光蛋白質)およびBSA(ウシ血清アルブミン))の正確な検出を中心に説明します。

As small soluble protein messengers released by cells, cytokines interact and communicate with other nearby cells. The results are pleiotropic and affect cellular function in ways that can indicate inflammation or disease progression, making cytokine detection, measurement, and monitoring important to clinicians and researchers alike. The enzyme-linked immunosorbent assay (ELISA) is the traditional way to detect and validate protein biomarkers in serum, cell preparations, or other biological sample types because the assay offers high target specificity.

In this application note, we compare Maurice™ against PerkinElmer’s LabChip® GXII Touch, a chip-based electrophoretic separation system. Under reduced and non-reduced conditions, we evaluate CE-SDS separation using a reference mAb from the National Institute of Standards and Technology (NIST). Maurice and LabChip are assessed for their performance on linearity, sensitivity, precision, reproducibility and resolution, with the technological approach, workflow and data quality outlined for easy comparison.

In this application note, we’ll focus on the 21 CFR Part 11 tools integrated into MFI for method and batch execution, data processing
and audit trails.

In this application note, we describe how you can generate great Single-Cell Western data with an Innoscan 710 scanner, which has the best combination of sensitivity, scan time and resolution needed to scan scWest chips.

In this application note, we’ll hone in on the 21 CFR Part 11 tools integrated into Compass for iCE for batch execution and data processing, plus audit trails and electronic signatures.

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.

In this application note, we demonstrate data equivalency across iCE instruments by running multiple molecules across all three systems. Data equivalency between iCE280 and iCE3 systems using Alcott and PrinCE autosamplers has been demonstrated before, so we focused on comparing system quantitation and reproducibility using absorbance mode on iCE280-PrinCE, iCE3-PrinCE, and Maurice systems.

In this application note, we compare endogenous PD-L1 detection in PTEN mutated glioma cell supernatant and blood cells using the Simple Plex assay and a commercially available sandwich ELISA assay to demonstrate data equivalency and the added sensitivity you'll get with a Simple Plex assay.

In this application note, we compare the data collected on Maurice using both hand-mixed samples and samples mixed using the on-board feature.

Every cell is unique, making cell-to-cell heterogeneity important in many areas of biomedicine including cancer pathogenesis, immuno-oncology and regenerative medicine. More and more high-profile publications are using single-cell analysis techniques to reveal variability in cellular response to a drug or stimulus. They're also uncovering variation in drug target expression within a tissue and identifying important subpopulations of cells within complex samples that play key roles in disease progression. Single-cell protein expression information is critical when you need to understand the fundamental composition and behavior of complex biological samples.

Milo, the first and only Single-Cell Western platform out there, lets you do Western blotting at the single-cell level. Now you can run Westerns on thousands of individual cells in parallel and get robust, Western-based information on protein expression heterogeneity in your cells. He also multiplexes so you can measure multiple proteins in each single cell. That means you can get a better understanding of correlations between target expression and characterize cell signaling in specific target-positive subpopulations of cells. Where your target is located in a cell doesn't matter to Milo — his fast, simple workflow lets you measure proteins both on and in each individual cell with the same workflow. It's easy to detect surface proteins and you don't have to worry about fixing and permeabilizing your sample to measure intracellular proteins. As an added bonus, Milo uses conventional Western antibodies which means you can measure diverse protein targets — even ones that don't have good flow cytometry antibodies. The best part? Scout™ Software automates your data analysis and gives you quantitative protein expression measurements in each single cell.

Mauriceの画像キャピラリー等電点電気泳動（cIEF）のネイティブ蛍光検出は、バックグラウンドが非常に低く、UVの吸収による検出に比べて感度が3〜5倍高いので、UV照射で検出する方法に比べ、より低い濃度のサンプルを用いて電荷不均一性分析を行うことができます。このアプリケーションノートでは、タンパク質が収束するにつれてタンパク質濃度が高濃度になり、沈殿・凝集しやすいタンパク質を用いてcIEFを行っても、ネイティブ蛍光検出することで、可溶化剤として知られる尿素濃度を減らす、もしくは完全に取り除いてもタンパク質は沈殿・凝集することなくアッセイを行うことができることを示します。

Milo enables scientists to perform single-cell resolution Westerns (scWesterns) for over 1,000 individual cells simultaneously, and in a fraction of the time of conventional Westerns. Researchers can now gain selective protein expression information for up to four protein targets in each cell, offering views into cell-to-cell variation within a complex sample.

iCE3 is the next-generation iCE280 and features a number of technical improvements for better system fluidics, systemto-
system reproducibility, and improved 21 CFR Part 11 options. All improvements are designed for direct transfer of
methods from iCE280. As a direct replacement for the iCE280 system it was a requirement that iCE3 and iCE280 have
equivalent applications performance. This document demonstrates system performance of iCE3 in comparison to iCE280
for all system configurations. The comparability experiments were performed using the following instruments and assays.

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

Peggy 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.

Unlike chemically synthesized drugs, protein therapeutics are a dynamic heterogeneous
mix of active compounds1. Due to their complexity, analytical techniques like isoelectric
focusing have become indispensable tools in evaluating biologic preparations. The
resulting surge in charge isoform analysis has led to major advances in instrumentation,
such as Imaged Capillary Electrophoresis (iCE�)2 . However, to obtain the full benefit from
improved instrumentation requires the coinciding development of robust assays.
Initially implemented in biopharmaceutical manufacturing, the holistic process
characterization philosophy known as Quality by Design (QbD) has the potential to
transform assay development3, 4, 5. Proper adaptation of these techniques will provide a
tremendous benefit to the robustness and predictability of assay performance. Key to
QbD is comprehensively gauging the effects of process inputs on critical to quality (CTQ)
attributes of the output3. To this end, the Design of Experiments (DOE) methodology has
proven itself to be a highly efficient tool in modeling the relationship between input and
output. Though statistical analysis packages such as SAS JMP and Minitab� have lowered
the computational barriers to executing DOE, generating meaningful results still requires a
working knowledge of the model building process.
The goal of this note is to promote the successful application of DOE tools in the assay development process by
offering a stepwise example. The road map contained in the following pages has purposely captured enough technical
detail to provide a comprehensive reference guide for both the statistician and analytical biochemist. The subjects that
will be covered include initial factor screening, construction of a central composite DOE, response surface modeling,
assay optimization, model validation and assay performance.

Three major usability improvements are now available for the iCE3 system. The new HT Cartridge improves resolution and run times by eliminating the need for methyl cellulose, saving up to 5 minutes per run when compared to the original FC cIEF Cartridge. Redesigned locking electrode arm hardware also reduces evaporation and minimizes cathodic drift, and updated software features allow automated pI calibration and data export.

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.

This document details the benefits and advantages of the Maurice system compared to iCE3.

This document supports the use of Micro-Flow Imaging® systems (MFI 5100, MFI 5200) in a QC and GMP environment. All functionality is integrated in MVSS (V5.0) for compliance with 21 CFR Part 11. The information presented assumes that the appropriate system configuration parameters have been set.

MFI Image Analysis software is a new tool suite for analyzing the particle images you’ve captured with Micro-Flow Imaging (MFI). MFI Image Analysis software improves your workflows and gives you a new set of user interface options for analyzing all the data you get from your MFI 5000 series.

iCE consumables make operating your system fast and
easy � and get you to more consistent data at the same
time. Our kits help you develop methods and run system
qualifications, and come with all the reagents you need
to simplify day-to-day operation. They�re also designed
to work with our FC Cartridges (P/N 101700, 101701),
which need methyl cellulose in both the rinse and sample
solutions to reduce sample interactions with the capillary.
GMP/GLP needs? All consumables are tested and validated
to meet them!

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.

Analysis with the iCE3 system just got faster! The new HT Cartridge reduces run times by 30% on the iCE3 with comparable results to the FC Cartridge. Its all-new, proprietary capillary coating eliminates the need for methyl cellulose in sample mixtures, which speeds sample loading and cuts focusing time - so runs that used to take 15 minutes are now done in 10. The HT Cartridge was developed and validated for the iCE3 and optimized for use with the iCE3 HT Upgrade.

The iCE3 with PrinCE Next Autosampler is an update to the popular iCE280 with PrinCE Microinjector system. This configuration offers even easier operation with no system balancing required. The PrinCE Next provides improved sample temperature control with new 50-vial sample tray and 96-well plate options. The iCE with PrinCE Next Autosampler also allows seamless method transfer between the iCE280 and the iCE3 systems.

Simple Imaging Analysis - Digital Darkroom
Get the right picture, right away with the protocol-driven touchscreen interface only available on Simple Imagers running Digital Darkroom.

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.

The best designed biologics manufacturing processes are efficient and reproducible. Streamlining the many steps in process development translates to less time required to get your product to market. We’ll help you test all your samples during development to save you time and money downstream. We’ll also help you confirm the integrity of your final products.

Protein analysis plays an important role in stem cell research, and for most researchers, Western blotting is a key component of their toolbox. From verifying pluripotency and identifying lineage-specific cell types to identifying key modulators of cell signaling pathways, to evaluating disease models and therapeutic approaches, the right protein analysis
techniques can help you achieve success. In this eBook, we examine technological advances in Western blotting and protein analysis that are at the forefront of stem cell research. Learn how automated Western systems and Single-Cell Westerns can advance your stem cell research.

Robust and fast analytical techniques are critical for achieving commercial success of therapeutic biologics and achieving it first. Speed up the development process with iCE and MFI from ProteinSimple. These highly sensitive technologies will streamline each step in the biologics characterization and manufacturing process so that you can win the race to FDA approval.

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.

Simple Western instruments further CRISPR/Cas9 applications by providing phenotypic validation at the protein level. Learn about this immunoassay technology.

By switching my assays over to Simple Western, Dr. Joseph Klebba is able to increase his throughput, allowing for full dose responses to be generated in each experiment. Utilizing the RePlex module even further increases this throughput as he often probes for 5-6 proteins per capillary in a single run including phosphorylated/total protein pairs in the same lane.

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

Ella Promises Better Clinical Care for Acute Respiratory Failure and Sepsis

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

Prof. Dr. Hermann Wätzig discusses the benefits of capillary electrophoresis and the automated solutions in the modern laboratory

The Global Forum on Childhood Pneumonia estimates that 11 million pediatric lives will be lost by the year 2030 if more global action is not devoted to accelerating research efforts against this disease. Among the actions required, is a commitment to research partnerships and breakthrough innovations to both advance our understanding of the immune response during infection and identify earlier time points for therapeutic intervention. Read on to learn how a team of clinician-scientists at Penn State Health are working to address this challenge using Simple Plex assays on Ella.

“With Milo, we observed a level of single-cell protein expression heterogeneity in our purified intestinal stem cell population that was not previously possible with conventional Western blotting.”

From Your Peers: Guts and Glory: Dr. Kaelberer Validates a Neuroepithelial Circuit using Milo

“[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."

“Wes is easy to use, convenient, and gives answers quickly. The throughput and system automation is a definite advantage.”

— Jane Gray, Ph.D., Head of Research Instrumentation, Cancer Research UK Cambridge Institute

“MFI technology has allowed us to better understand what conditions promote stability of our therapeutic molecules, which ensures that only world-class medicines are delivered to patients.”

— Stephanie Davies, Ph.D., Formulation Sciences, MedImmune

"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 no-gel, no-transfer, no-membrane features are beneficial for the reproducibility of the assay. I really like this system over traditional Western."
— Kazuya Machida, M.D., Ph.D., Associate Professor, Department of Genetics and Genome Sciences, UConn Health

“Simple Plex assays on Ella gave us speed and efficiency so we could complete our assays quickly, and the high quality of the data meant that we could easily see differences between disease state samples and controls. Plus, sample preparation was so simple it reduced chances of errors significantly.”
— Paulomi Aldo, Research Associate and Reproductive Sciences Core Manager, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University

"We had been trying to study receptor proteins with traditional Westerns but it was frustrating — we just could not get it. A few months with Wes and we've already had way more success than we had in three years."
— Brian Perrino, Ph.D., Associate Professor, Department of Physiology and Cell Biology, University of Nevada School of Medicine

”We collect samples from laser microdissection and were using our entire sample on just one regular Western blot. With Wes, we can do multiple assays with one sample collection.“
— Mary Howell, Laboratory Coordinator, Department of Internal Medicine, East Tennessee State University

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.

Simply Speaking Newsletter, volume 3, issue 2

Simply Speaking Newsletter, volume 2, issue 3

Simply Speaking Newsletter, volume 2, issue 1

Simply Speaking Newsletter, volume 1, issue 2

このポスターではMaurice CE-SDSの分子量範囲（10～270 kDa）をどのように広げ、小さいタンパク質や巨大タンパク質を分析することができるのかを示します。

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。

Therapeutic monoclonal antibodies (mAbs) make up a large portion of the rapidly growing drug market. Ensuring safety and efficacy through comprehensive understanding of these products’ critical quality attributes (CQAs), including charge heterogeneity, is a regulatory requirement. Various charge isoforms of mAbs can result from cell culture or production processes, potentially affecting the mAb structure and function. While imaged capillary isoelectric focusing (icIEF) is the preferred method for charge profiling, ion-exchange chromatography (IEC) has been the major tool for fractionation combined with characterization. However, IEC is not compatible with certain types of molecules, hydrophobic antibody drug conjugated (ADCs) for example, and icIEF typically provides higher separation resolution. Moreover, an individual charge variant obtained from IEC fractionation may not be comparable to the variant peak in the icIEF profile. Therefore, there is an unmet need for IEF-based fractionation of charge variants for characterization.
We have developed a novel icIEF fractionation solution, which involves icIEF separation and collection of charge variants. This solution enables Maurice icIEF-based peak identification followed by downstream analysis. Here we report icIEF fractionation followed by ZipChip-based mass spectrometry (MS) characterization of the NIST mAb and XMT-1535 mAb. ZipChip (CE-ESI) was utilized for mass spectrometry characterization of the fractions due to its broad sample matrix compatibility, easy sample prep, and fast mass spectrometry analysis time. Individual charge variants of each antibody were successfully collected in less than 2 hours with purity > 80% using icIEF separation conditions with or without urea. Rapid analysis using ZipChip chowed the mass spec identification of major and minor isoforms correlated well with reported mass spec data (literature and report). Urea in icIEF separation did not affect the quality of fractionation nor the mass spec result. Multiple fractionation runs of the NIST mAb suggested good reproducibility of the system. We believe this novel icIEF fractionation solution coupled with other analysis methods, such as mass spectrometer, will deliver a powerful charge variant characterization tool for biotherapeutic analytical tool kit.

icIEF is well-established as the gold standard tool in the biopharmaceutical industry for protein charge characterization. Newer therapeutic modalities are coming to market, including viruses and virus like particles. A challenge associated with icIEF analysis of virus and virus like particle (VLP) samples is sample aggregation during IEF. For most sample (mAbs, proteins), adding solubilizers into the sample solution, such as urea and non-iconic surfactants, can prevent aggregation. However, intact viruses and VLPs may disassociate when these additives are used. While reducing the final sample concentration can help minimize aggregation, UV light absorbance-based detection may not possess sufficient sensitivity to analyze these samples at lower concentrations. In the presentation, we will illustrate the results of using an icIEF-UV fluorescence instrument for charge characterization of viruses and VLP samples. The UV fluorescence method requires no dye labeling and shows significantly higher sensitivity than UV absorption detection.

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.

Adeno-associated viruses (AAV) are promising vectors for the delivery of genetic material in gene therapy. During the manufacture of AAV, critical quality attributes like charge heterogeneity and purity must be carefully monitored because they can impact the product’s safety and efficacy. Imaged capillary isoelectric focusing (icIEF) and capillary electrophoresis sodium dodecyl sulfate (CE-SDS) are two powerful methods to respectively characterize charge heterogeneity and purity, but traditionally two separate platforms are required to run these methods. Here, we used a single platform to develop icIEF and CE-SDS methods to analyze AAV2 and AAV6 serotypes to monitor product stability, identity and purity. We show that these methods could reproducibly quantify both intact (by icIEF) and denatured AAV (by icIEF and CE-SDS) samples. The CE-SDS method could separate and quantify individual AAV capsid proteins, showed robust repeatability (<5% RSD) while also detecting impurities. The icIEF method was useful for measuring both denatured and intact particles with high repeatability (<4% RSD). Interestingly, preliminary evidence suggests that icIEF can also distinguish between full (1 x 10^13 GC/mL) and ‘empty’ (<10^12 GC/mL) AAV capsids.

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.

CRISPR/Cas9 has become a revolutionary tool for precise genome editing in a wide variety of prokaryotes and eukaryotes, and multiple Cas9 variants have been engineered to further broaden its functionality. Purity, heterogeneity and stability are critical properties in the biophysical characterization, chemical modification and structural investigation of Cas9 and its variants. At the forefront of techniques to monitor these properties are capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and capillary isoelectric focusing (cIEF). Maurice™ from ProteinSimple enables both CESDS and imaged cIEF (icIEF) in a single unit, with several key advantages over other CE-SDS and cIEF systems, such as ease-of-use, high data quality, and speed.

Axonal damage is a hallmark of neurodegenerative disorders. In various neuropathologies, injured or degenerated axons release their intracellular components into the extracellular space, resulting in circulation of these molecules in the cerebrospinal fluid (CSF) and peripheral blood. Consequently, monitoring the levels of these molecules in biological fluids can serve as a potent biomarker for the diagnosis, prognosis, and evaluation of treatment in neurodegenerative diseases.

キャピラリー等電点電気泳動（IEF）分析中において、尿素は一般的にタンパク質可溶化剤として使用され、タンパク質の凝集や沈殿を軽減します。このポスターでは、尿素の代替え試薬として弊社の提供するSimpleSolはpH勾配の酸性部分への影響が少なく、IEFのタンパク質を効果的に可溶化でき、尿素よりもはるかに安定した結果をもたらすことを示します。毎回フレッシュな尿素を調製する必要がないことを示します。尿素には、タンパク質のカルバミル化を引き起こす可能性があり、毎回フレッシュな尿素溶液を調整する必要がありますが、溶液として提供されるSimpleSolは溶液の安定性、pH勾配の形成、および使いやすさの利点により分析ラボにとって日常的に使われる有用な添加剤として位置付けられます。

In this poster we show that this reagent, SimpleSol can
effectively solubilize proteins for icIEF but is a significantly more stable agent than urea, eliminating the need for analysts to prepare urea fresh every time SimpleSol is also stable when pre mixed with methylcellulose as opposed to urea and is compatible with absorbance and native fluorescence detection on Maurice icIEF In addition we show that SimpleSol had less of an impact on the acidic portion of the pH gradient formed during icIEF compared to urea, resulting in more stable pI values of protein peaks relative to using urea.

We designed a quantitative multi-analyte detection assay of key cytokine release syndrome (CRS) biomarkers. This microfluidic parallel immunoassay measured four cytokines simultaneously in only one hour. We compared cytokine measurements for human IL-6, IL-1β, TNF-α, and IFN-γ between our Quantikine® ELISA and Simple Plex™ immunoassays using an in vitro model for CRS.

The goal of personalized medicine is to stratify individual patients to the appropriate treatment. This approach depends on extensive characterization of individual tumors and their sensitivity to therapeutics. In the context of the immunotherapy of cancer, information on the localization, abundance and activation of immune cells within individual tumors gained in importance. Here we present a preclinical drug testing model to monitor individual drug responses of patients to targeted immunotherapy with the checkpoint inhibitor Nivolumab (anti- PD-1) and subsequent applications. In this study, we were able to determine different populations of infiltrating immune cells within viable tumors from colorectal cancer patients using our drug testing platform and a variety of subsequent applications.

Analysis of immune cells was conducted on disaggregated cells from viable tumor slices. Disaggregation of precision cut cancer tissue slices was performed using the GentleMACS from Miltenyi. Immune cell subsets were analyzed by flow cytometric multiplexing of CD3, CD4, CD8 and CD45. Furthermore, we identified PD-1 positive cells among the CD45+/CD3+ lymphocyte population, indicating relevance for anti-PD-1 targeted therapy in colorectal cancer. Presence and localization of immune cells (CD45+) was confirmed by immunohistochemistry of tumor tissue slices. In addition, protein expression of CD8 and PD-1 was analyzed by Simple Western Size analyses. Cytokine secretion affected by Nivolumab treatment was analyzed in supernatants of tissue cultures using the proinflammatory panel from Meso Scale Discovery. The results demonstrated that immune cell compositions were stable and uniform within our precision cut cancer tissue slices both pre- and post-cultivation, and pre- and post-treatment with Nivolumab.

AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and has been revealed as the most highly expressed gene in preclinical models with acquired resistance, and second most common alteration in EGFR (epidermal growth factor receptor) inhibitor-resistant tumors, behind the T790M mutation. It has become obvious that targeted therapy of patients has to be monitored by taking and analyzing biopsies on a regular basis. Therefore, laboratory methods have to be adapted.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis.

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.

Monoclonal antibodies (mAbs) are routinely used as therapeutic and diagnostic products when treating a wide variety of diseases. Due to the importance of these molecules, regulatory agencies have guidelines describing methods to assess product purity necessary for lot release and characterization. CE-SDS is one of the most common techniques used to monitor purity and heterogeneity.

iCE3 has been the go-to method for analyzing charge heterogeneity for mAbs. In this poster, we introduce Maurice, the latest member of the iCE platform family, who now gives you high resolution CE-SDS IgG data on top of the exceptional cIEF data you've come to expect.

Maurice's CE-SDS application gives you baseline resolution of reduced non-glycosylated and glycosylated IgG heavy chain in just 25 minutes with % Area RSDs less than 4%. A simplified workflow provides unparalleled ease-of-use. Once samples and reagents are prepared, it takes less than 10 minutes to install the cartridge and start your batch. And at the end of your batch, easy clean-up and automatic data analysis by Compass for iCE software lets you quickly start your next batch.

Traumatic brain injury (TBI) affects up to 10 million people worldwide. Mild traumatic brain injury (mTBI) accounts for between 70-90% of all TBI cases. An estimated 10-20% of all veterans of recent U.S military conflicts have sustained mTBI. The primary goal of TBI biomarker research is to identify molecular changes in the brain that could help determine if the brain was injured and help monitor the recovery process.

Proinfl ammatory biomarkers are released following brain injury and induce a neuroinflammatory response. The prolonged presence of these biomarkers can affect neurons and brain functioning. Inflammation can also alter cell phenotypes and cause increased cytokine production and neural damage. A single blood inflammatory biomarker may not indicate brain pathology; however, evaluating multiple biomarkers may be more informative and allow for a more accurate diagnosis. We evaluated multiple neuroinflammatory markers using Simple Plex™, a novel quantitative, multianalyte immunoassay platform that delivers high precision and accuracy with ≤ 25 uL of sample. Our results identify a potential inflammatory profile for TBI.

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.

Concentration and morphologic distributions of particle sub-populations is required to ensure product quality of biopharmaceuticals. However, current pharmacopeial methods are not optimized for transparent protein aggregates which can lead to undercounting. MFI addresses this technology gap, combining sensitive image capture and morphologic analysis that can discriminate translucent protein particles from silicone micro-droplets and other particles.

In order to bring an antibody or other biologic drug to market, each step of the process needs to be carefully monitored. Charge heterogeneity and apparent molecular weight (MW) via SDS-PAGE are two of the most commonly assessed parameters. Ideally, similar methods should be used from the beginning of development (low expression, complex matrix) to late in production and QC (high concentration, pure material). Capillary techniques, such as iCE and CE-SDS, are currently used heavily in downstream product development but are best suited to purified and higher concentration samples. Here, we present the Simple Western that combines in one novel instrument, Peggy, capillary electrophoresis with an immunoassay to provide highly reproducible and fully automated analysis of monoclonal antibodies. This sensitive technology measures either size or charge in complex samples and provides critical charge heterogeneity, size, and product titer information without the need for sample purification. Data will be presented demonstrating the application of the Simple Western technique and the iCE technology for the analysis of monoclonal antibodies against VEGF and the ability of the two techniques to provide consistency of data across the whole range of product development. In addition we show examples for the unique capability of this technology to assess affinity information for these anti VEGF antibodies to different charge isoforms of VEGF.

Three major usability improvements are now available for the iCE3 system. A new HT cIEF Cartridge increases resolution while reducing run times by eliminating the need for methyl cellulose. A redesigned electrode arm assembly minimizes cathodic drift allowing robust analysis of 100 samples in a batch. The pI calibration and data export processes have been combined into a single automated procedure through the development of enhanced software features. These updates offer greater speed and improved ease of use. In this poster we present the results of these improvements on a model iCE method for analysis of a basic IgG1 mAb. The new HT cartridge reduces analysis time by five minutes, while still providing a highly resolved peak profile comparable to the original FC cartridge. An intermediate precision study demonstrated a %CV of less than 10% for peak all major peak clusters (>5% percent composition).

iCE3 Separations
Separations were performed on an iCE3 system
equipped with either a PrinCE Next MicroInjector or an
Alcott 720 NV Autosampler.

Analytical techniques like isoelectric focusing have become indispensable tools in evaluating biologic preparations. The resulting surge in charge isoform analysis has led to major advances in instrumentation, such as Imaged Capillary Electrophoresis iCE�. To maximize the benefits from improved instrumentation requires development of robust assays. Initially implemented in biopharmaceutical manufacturing, Quality by Design (QbD) has the potential to transform assay development. Key to QbD is comprehensively gauging the effects of process inputs on critical to quality (CTQ) attributes of the output. To this end, the Design of Experiments (DOE) methodology has proven itself to be a highly efficient tool in modeling the relationship between input and output.
The contents of this poster demonstrate the successful implementation of DOE tools in the iCE3 assay development process. Utilizing a Central Composite Design (CCD) strategy, a response surface of the relationship between ampholyte composition and focusing time on peak resolution was generated. Experimental validation of this model at optimal operational settings indicated a high level of accuracy with error between the predicted and experimentally derived values ranging between -0.68 and 8.06 percent.

Many features of protein-based pharmaceuticals limit the ability of standard particle analysis methods to characterize them. For example, high particle concentrations, heterogeneous particle types, viscosity, and a low refractive index are known to reduce the detection and sizing accuracy of light obscuration and membrane microscopy. As reviewed in this poster, Micro-Flow Imaging (MFI) technology can analyze many types of challenging protein samples using direct, imaging-based particle measurement. Particle types detected include semi-transparent protein fragments, air bubbles, and contaminants such as silicone oil micro-droplets. Moreover, MFI detection is largely independent of a particle's optical properties; thus it can handle concentrated antibody solutions, as well as viscous samples.

Proteins in the AKT signaling cascade are associated with tumor cell survival, proliferation, and invasiveness. The activation of AKT is also one of the most frequent alterations observed in human cancer and tumor cells. Understanding how AKT signaling impacts disease progression is important to the development of novel therapeutics. Cell signaling events are routinely assessed using traditional Western blot analysis. The Western blot technique is very labor intensive and generally yields results that are semi-quantitative. The Simple Western platform described here completely automates the manual steps involved in traditional Western blot protocols and can analyze up to 96 samples in a single experiment. Because Simple Western protocols consume only microliter sample volumes, reproducible and quantitative results can be generated from precious or quantity-limited samples.

We present results generated on Sally, the higher throughput platform from ProteinSimple. Sally is able to run up to 96 data points in a single experiment, addressing the needs for higher throughput applications in screening signaling pathways. Data generated on Sally demonstrates high reproducibility and intra-assay variability of less than 10%.

Seven targets from the AKT pathway, were screened with both anti -total and anti-phospho antibodies on the Simple Western platform from a total volume of 5 �L/treatment. In response to Insulin treatment, significant and expected signal changes were clearly observed for each of the key targets. This suggests the potential to characterize whole signaling pathways with as little as 5 �L of sample. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods.

When methods and instruments are implemented in Quality Control, it is critical that any replacement instrument provide equivalent data with minimal or no changes to the method. The iCE280 system has been adopted by most major biopharmaceutical companies worldwide for charge heterogeneity analysis. The iCE3 is the next-generation iCE280 and the first update to the iCE280 in 15 years. The iCE3 features a host of technical improvements, all designed for direct transfer of methods from the iCE280. As a direct replacement for the iCE280, the iCE3 must have equivalent applications performance. In this poster, we demonstrate direct method transfer and system equivalency using the following instruments and assays.

Capillary isoelectric focusing (cIEF) is the best tool for protein charge heterogeneity characterization. Common sources of charge heterogeneity for monoclonal antibodies include heavy chain C-terminal lysine heterogeneity, deamidation, and sialyation associated with the glycosylation sites on the antibodies. Under the denatured and reduced condition, disulfide bonds in the antibodies can be reduced and the monoclonal antibodies are broken into heavy chains and light chains. The heavy chain and light chain are expected to have different pI values, thus, they can be separated from each other by IEF. In this way, the contributions of the heavy chain and light chain to the entire antibodies' charge heterogeneity can be observed by IEF analysis.

In this presentation, monoclonal antibodies are analyzed by cIEF under a 8 M urea (denatured) and DTT condition. In the example shown in the presentation, the heavy chain and light chains are well separated and the charge heterogeneity of both is observed.

During capillary isoelectric focusing (cIEF), proteins are focused at their isoelectric point. When performing IEF in a capillary, two forces within the capillary can destroy the IEF process within the capillary column: electroosmotic flow (EOF) and hydrodynamic flow. For reproducible cIEF, both of these forces must be eliminated. EOF is suppressed by neutral coatings on the inner wall of the capillary. Elimination of hydrodynamic flow is dependent on the system design. Traditional capillary electrophoresis instruments minimize hydrodynamic flow by leveling the capillary inlet and outlet vials. In imaged cIEF, specifically the iCE280, elimination of hydrodynamic flow depends on the autosampler. Small differences in hydrodynamic flow can result in differences in reported pI values from system to system. Although the difference is relatively small, it may create issues during method transfer between labs.

In this poster, we describe how the new iCE3 IEF Analyzer simplifies method transfer and minimizes system-to-system variability caused by hydrodynamic flow.

The well-characterized biopharmaceutical requires an assessment of charge heterogeneity. Techniques like IEF gels, ion exchange chromatography, and traditional capillary IEF all have benefits, but each one has its own set of challenges. Imaged cIEF (ProteinSimple's iCE IEF Analyzer) combines the best of these three worlds by providing rapid analysis, platform methods, and simple method development.

For cIEF analysis, samples are pre-mixed with carrier ampholytes, pI markers, and other additives. Although iCE IEF Analyzer offers rapid analysis and high throughput, some proteins can still experience degradation when exposed to these conditions for extended periods. On-Board Sample Preparation with the new iCE3 IEF Analyzer solves this problem. The system prepares the sample immediately prior to injection, limiting sample exposure to cIEF buffers and preventing degradation. As an additional benefit, automated sample preparation eliminates tedious pipetting. Simply load your sample and go.

This poster presents the application of On-Board Sample Preparation to the analysis of proteins.

The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly negatively correlated with L-ASP anticancer activity in ovarian cancer cell lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. However, quantitatively robust, single-antibody assays for ASNS expression have been absent from the literature. We therefore used a capillary-based isoelectric focusing (IEF) platform (the NanoPro 1000) to screen twelve ASNS antibodies for their specificity and sensitivity. Only two antibodies exhibited completely on-target activity (as shown by signal ablation by ASNS siRNA) and sufficient sensitivity. The on-target activity corresponded to a single band on Western blot and a single peak on the NanoPro 1000, suggesting the existence of just one ASNS protein isoform. Optimized, final NanoPro assay conditions yielded less than 8% CV, a 160-fold dynamic range, and Z′-factor of 0.82, indicating a robust assay that is amenable to high-throughput screening. We next used the best ASNS antibody to develop an immunohistochemistry (IHC) assay for ASNS. As with the NanoPro assay, optimized IHC conditions yielded a large dynamic range of staining intensity, and staining was completely ablated by ASNS siRNA. To test the hypothesis that subsets of various cancer types express very low levels of ASNS, we have initiated ASNS IHC of more than 20 tissue arrays representing a wide variety of cancer types. Using a 3-point scoring system (0 = negative, 1 = low, 2 = high), among the tumor samples assayed, 90/136 (66%) of bladder cancer, 63/133 (47%) of bone cancer, 32/149 (22%) of breast cancer, 29/115 (25%) of brain cancer, 51/168 (30%) of colon cancer, 2/85 (2%) of endocrine system cancer, 23/99 (23%) of liver cancer, 7/64 (11%) of head and neck cancer, 7/136 (5%) of lung cancer, 13/53 (25%) of lymphoma, 1/25 (4%) of bone marrow lymphoma, 2/35 (6%) of lymphoma from spleen, 9/109 (8%) of melanoma, 81/396 (21%) of ovarian cancer, 3/29 (10%) of uterine cancer, 27/73 (37%) of pancreatic cancer, 5/119 (4%) of prostate cancer, 10/125 (8%) of renal cancer, 25/138 (18%) of testicular cancer, and 8/39 (21%) of thyroid cancer were ASNS-negative (score = 0), suggesting that a subset of each cancer type may be sensitive to the drug L-asparaginase. Efforts are underway to apply the NanoPro assay to the NCI-60 cell line panel and to continue performing ASNS IHC to survey tissue arrays for ASNS expression.

Novel inhibitors of the hypoxia pathway [VEGF, PDGF] achieve response rates of 30-57% in renal cell carcinoma (RCC); yet threshold levels of targets and downstream signaling proteins have not been identified as biomarkers to guide treatment.

Methods: To profile hypoxia proteins in RCC clinical specimens, we have developed the use of automated nanoscale immunoassays for charge-based protein separation (NIA, NanoPro 1000) and charge-based
protein separation (Simple Western, Sally). To decrease the amount of tissue and invasive procedures required to obtain cells for analysis, we optimized assays to profile specimens acquired by fine needle aspiration (FNA).

Results: We used Simple Western to quantify proteins of the MAPK (ERK1, ERK2, pERK1, pERK2, MEK2), PI3K (S6, GSK3b, AKT2, pan-AKT) and STAT pathways (p-STAT5) and loading controls (tubulin, HSP-70) in more than 200 FNA's from solid tumors including RCC. Profiles can be completed overnight after receiving the specimen. Unique to NIA, we also analyzed percent phosphorylation and resolved differences in even a single phosphorylation in FNA specimens, allowing us to group tumors based upon different patterns of phosphorylation and percent phosphorylation.

Conclusions: Rapid and quantitative nanoproteomic profiling in very small amounts of clinical specimen is enabling translational studies for novel diagnostic and predictive biomarkers.

Aberrant expression and signaling of multiple proteins in the NF-κB pathway are commonly associated with inflammatory and stress-induced diseases, including many cancers. Understanding how NF-κB signaling impacts disease progression is important to the development of novel therapeutics. Cell signaling events are routinely assessed using traditional Western blot analysis. The Western blot technique is very labor intensive and generally yields results that are semi-quantitative. The Simple Western platform described here completely automates the manual steps involved in traditional Western blot protocols and can analyze up to 96 samples in a single experiment. Because Simple Western protocols consume only microliter sample volumes, reproducible and quantitative results can be generated from precious or quantity-limited samples.

We present, for the first time, results generated on Sally, the newest member of the Simple Western platform from ProteinSimple. Sally is easy to set-up and runs hands-free up to 96 data points in a single experiment, thus addressing the need for higher throughput. Sally generates 8 individual measures from 5 µL of sample which allows for characterization of whole signaling pathways from one small sample size.

Data generated on Sally from examination of targets in the NF-κB (p100/p50) pathway demonstrate high reproducibility and low intra-assay variability. Response to TNF-α treatment in whole cell and nuclear extracts of IκB and NF-κB subunits (c-Rel, p65, and p50/p105) demonstrates, as expected, statistically significant changes in signal and localization. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods.

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.

Charge-based separation of peptides prior to LC-MS analysis has been hampered by long run times and the presence of components such as detergents and carrier ampholytes that interfere with MS (1). Cell Biosciences has developed a workflow using the digital ProteomeChip� (dPC�), that captures peptides in acrylamide gel plugs according to charge, with run times less than one hour (2,3) (figure 1). This workflow employs buffers and conditions that have been designed to be �MS-friendly,� eliminating the need for postseparation sample clean-up and dramatically reducing artifacts introduced by the separation processes. Cell Biosciences ProteomeChips are available in three pH ranges: 3.5-4.5, 4.2-6.2 and 6.0-8.0, providing an overall pH range of 3.5-8.0.

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.

Oncoprotein quantification in clinical specimens is important for the diagnosis of specific hematopoietic malignancies as well as the development and monitoring of effective therapies that target oncoproteins. Current protein detection methods require large samples, precluding routine serial tumor sampling to assess changes in oncoprotein levels. Here we demonstrate the use of a nano-immunoassay system (Firefly™ system) to distinguish between patient specimens of Burkitt's vs. Follicular lymphoma by characterizing patterns of MYC and BCL2 expression. Changes in the expression and activation of a variety of onco/signaling proteins including ERK, MEK, STAT and JNK in malignant hematopoietic (CML) cells and patient samples before and after treatment with therapeutic agents that impact oncogenic signaling pathways are also shown. The expression levels of the different proteins were measured with high sensitivity in samples as small as 400 cells. A key benefit of this technology is that it separates protein isoforms based on its isoelectric point. Using this assay, we were able to distinguish and quantify the phosphorylated and non-phosphorylated forms of each protein with a single antibody. Thus, we have developed a novel technique which can precisely evaluate the activity levels of signaling proteins in oncogenic pathways from very small samples.

The discovery of tumor stem cells in acute myeloid leukemia a decade ago initiated a field of research has accelerated growth in the past few years. Researchers are now describing tumor stem cells in a variety of hematopoietic and solid tumors. The impetus for much of this research is the desire to identify targets for drug intervention in these critical tumor populations. The molecular pathways that functionally define these cells are important therapeutic targets. Tumor stem cells are rare and provide insufficient material to use standard assay methods. Although DNA microarray and/or qPCR are used to study tumor stem cells, their rare nature limits quantitative protein analysis. This creates a gap in our knowledge since many proteins, such as beta-catenin or MAPK signaling proteins, are not regulated at the transcriptional level, but through post-translational modifications (phosphorylation, ubiquitination, etc.). Here we describe a technique utilizing a nanoimmunoassay platform (Firefly?) to measure tumor stem cell proteins. Transitional tumor stem cells (TCC+) were sorted from a patient tumor and lysed for analysis. A lysate of 400 cells was subjected to isoelectric focusing and immobilization. Immunodetection was performed and quantitation of signal was measured using HRP-labeled secondary chemiluminescence reagents. Here we report beta-catenin protein concentrations of 192 ng/mg of total protein in the tumor stem cells, which was undetectable in 'non-stem' tumor cells. Comparisons of protein levels and the degree of phosphorylation are made between these samples, other tumor cell lines and hematopoetic stem cells.

MauriceまたはMaurice Cシステムの電荷不均一性分析に使用するMaurice cIEFカートリッジの製品説明書。

CE-SDS plus application kitには、CE-SDSアプリケーションを行うのに必要なすべてのものが含まれており、1キットあたり200サンプル測定することができます。

MauriceまたはMaurice Cシステムの電荷不均一性分析に使用するMaurice cIEFカートリッジの製品説明書。

MauriceおよびMaurice CシステムでcIEFアプリケーションのパフォーマンスを8回確認することができるcIEF Chemical Test Kitの製品説明書。

CE-SDS application kitには、CE-SDSアプリケーションを行うのに必要なすべてのものが含まれており、1キットあたり200サンプル測定することができます。

Turbo CE-SDS application kitには、CE-SDSアプリケーションを行うのに必要なものがすべて含まれており、1キットで最大200サンプル分測定することができます。

製品説明書: Turbo CE-SDS Reagent Kit (PS-MRK01-TS)

Fluorescence Separation Moduleと一緒に、タンパク質分離を分子量で行うシンプルウェスタンを行うと、これらのDetection Moduleは、一次抗体で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。これらのDetection Moduleは、Jessでのみ使用することができます。

製品説明書: Fluorescence and Chemiluminescence Training Kit (PS-T005)

製品説明書: Chemiluminescence Training Kit (PS-T001)

製品説明書: Stellar Training Kit (PS-T007)

製品説明書: Total Protein Detection Module for Wes, Peggy Sue and Sally Sue (DM-TP01）

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

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

• List of Related Consumables

Product Insert: Jess Training Kit