Simple Western technical library

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Simple Western

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Application Notes

		SIMPLE WESTERNでCOVID-19マルチ抗原を応用した血清学的IgG抗体アッセイ \| Japanese	[概要表示]
		このアプリケーションノートでは、ProteinSimpleのSARS-CoV-2 Multi-Antigen Serology Module for Jess/Wesが、ヌクレオカプシドやスパイクタンパク質と同様にスパイクタンパク質のS1、S2およびS1RBDサブユニットなど、一般的にCOVID-19に関連する5つのウイルス抗原に反応するヒト血清IgGを同時に検出する方法を示します。これら5つの抗原に反応する様々なヒト血清IgGは、感染や中和作用の異なる段階を示している可能性がありますが、SARS-CoV-2に対する免疫応答を1度の迅速かつ簡単なアッセイでより詳細に理解します。各ウェルで1つの標的タンパク質を観察する主要な血清学的分析方法として知られるELISAやその他のアッセイでは、SARS-CoV-2に対する複雑な免疫応答を幅広く理解することが困難となります。
		SIMPLE WESTERNでリプロービングJessで行うRePlex！\| Japanese	[概要表示]
		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。
		創薬におけるターゲットエンゲージの評価: WesによるCellular Thermal Shift Assay \| Japanese	[概要表示]
		CETSAは、細胞内での薬剤のターゲット分子へのエンゲージを直接測定評価する方法で、その特異性も評価することができます。従来使われてきた方法では、代謝産物量の変化や、その下流分子におけるリン酸化状態、薬に対する細胞生存率を指標としてきていたため、薬とターゲット分子との相互作用を直接その表現型に結び付けることができませんでした。このアプリケーションでは、ProteinSimpleのWesを用いて優れた再現性で定量的CETSAデータを得ることを示します。薬剤とターゲット分子の結合を確認し、定量的性質や50%阻害濃度 (IC50)も算出します。
		Simple Westernで遺伝子・細胞治療用のアデノ随伴ウイルス（AAV）のタンパク質分析 \| Japanese	[概要表示]
		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。
		Simple Westernで血清中の抗体解析を簡素化 \| Japanese	[概要表示]
		このアプリケーションノートでは、Simple Westernを用いて全身性エリテマトーデス患者血清内の自己抗体を検出し、血清中の特定の抗体を検出し定量できることを証明します。Simple Westernアッセイはサンプルごとに独立したキャピラリー内で行われ、サンプルの分離からデータ分析まですべてが完全に自動化されています。メンブレンを短冊にカットし、洗浄とインキュベートをマニュアルで行い、検出前にそれら全ての短冊を分子量マーカーで並べたりする必要はもうありません。
		Simple Westernでマルチプレックス \| Japanese	[概要表示]
		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。
		Lentiviral Vector Analysis for Cell and Gene Therapy Made Simple	[概要表示]
		Simple Western is a capillary electrophoresis immunodetection platform that allows you to measure lentiviral vector identity, titer, stability, purity, capsid content, and transduction efficiency, all in one instrument.
		SIMPLE WESTERN JESSにSTELLAR NIRとIRモジュール登場 \| Japanese	[概要表示]
		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。
		AAV2 VP Protein Standards and Their Use in Quantifying Capsid Protein Ratio by Western Blot and Simple Western	[概要表示]
		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.
		Do Your AAVs Contain DNA? Rapid and Sensitive Empty/Full Capsid Quantification with Simple Western	[概要表示]
		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).
		Stellar NIR and IR Modules on Jess for Simple Western	[概要表示]
		Industry-leading fluorescence sensitivity for Western blot analysis
		Shining New Light on Pharmacokinetic Assays with Simple Western	[概要表示]
		Simple Western was used to separate proteins by charge and size to evaluate the pharmacokinetic properties of adalimumab and two adalimumab biosimilars in human serum.
		Keeping the Promise of Immuno-Oncology with Simple Western and Single-Cell Western	[概要表示]
		Simple Western and Single-Cell Western have the potential to make significant contributions to three important branches of immuno-oncology: angiogenesis of the tumor microenvironment, immune checkpoint inhibition, and chimeric antigen T-cell (CAR T-cell) therapy. Learn more.
		Concentrating on AAV Impurities with Ultrasensitive Total Protein Detection on Simple Western	[概要表示]
		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.
		Automated Profiling of PROTAC®-Induced Cereblon Neosubstrate Degradation Using Simple Western	[概要表示]
		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	[概要表示]
		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.
		Simple Western Assays for Detection of ACE2 and TMPRSS2, Key Players in SARS-CoV-2 Infection	[概要表示]
		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.
		Reprobe Your Immunoassay Samples Using RePlex with Jess	[概要表示]
		To gain the most data out of your precious sample, Jess provides a two-step immunoassay that is performed within the same capillary. This new feature, called RePlex removes the antibodies from the first round of probing to perform either a second round of probing with new antibodies or total protein detection. Importantly, RePlex efficiently removes antibodies between probing without compromising the integrity of the immobilized protein or its epitopes, allowing for excellent reproducibility across cycles. With Jess’s chemiluminescence and fluorescence channels, you can detect multiple targets per cycle. The second cycle can also be dedicated to total protein detection so that you can normalize your data with confidence. All the steps of RePlex are automatically performed with Jess, providing more data, and lowering the cost of reagents and consumables per sample.
		Wes and Milo Synergize to Profile Immune Cell Populations in the Tumor microenvironment	[概要表示]
		In this application note, we’ll show you how Wes™ and Milo™ partner to get you critical answers to 1) what type of immune cell populations are present in a sample and then 2) what percentage of cells in that sample make up a specific immune cell subtype.
		Simple Western Analysis of Adeno-Associated Virus (AAV) Proteins for Cell and Gene Therapy	[概要表示]
		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.
		WesとMiloの相乗効果で腫瘍微小環境の免疫細胞集団のプロファイルを作成 \| Japanese	[概要表示]
		腫瘍微小環境は、がん細胞や、非がん性および免疫細胞の統合ネットワークであり、その相互作用により腫瘍の不均一性、転移の広がり、そして後天的な薬剤耐性が促進されます。特に、腫瘍微小環境におけるリンパ球、マクロファージ、樹状細胞などの白血球の浸潤は重要な予後予測因子であり、がん免疫療法により期待される治療効果を妨害する主な障害要因としても認識されています。このアプリケーションノートでは、Wes™とMilo™がどのように連携して、まず、1）サンプルにはどのような種類の免疫細胞が存在するか、そして、2）何パーセントの細胞が特定の免疫細胞サブタイプなのかという重要な問いかけに答えるのかを示します。
		Multiplexed Western Blotting Redefined: Superplexing on Jess	[概要表示]
		In this application note, we simultaneously assess p-Akt and total Akt levels using different detection channels combined with in-capillary protein normalization to demonstrate the superplexing power Jess possesses.
		Simple Westernでバイオプロセス中の不純物質の検出 -医薬品製造過程関連の不純物の同定および定量- \| Japanese	[概要表示]
		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。
		Easy Transfer of Your Traditional Western Blot to Wes \| Japanese	[概要表示]
		このアプリケーションノートでは、どのように従来のウエスタンブロットアッセイからWesにアッセイを移行するかについて、その概要を説明します。
		High-throughput glycan characterization using Simple Western

		Bioprocess Contaminant Detection using Simple Western	[概要表示]
		In this application note, we focus on the accurate detection of four candidate contaminants that may be present during various stages of the therapeutic protein and vaccine development processes: host cell protein (HCP), Protein A, green fluorescence protein (GFP) and bovine serum albumin (BSA).
		Monitoring Target Engagement in Drug Discovery: Application of Wes to the Cellular Thermal Shift Assay	[概要表示]
		In drug discovery, confirmation of in-cell target engagement is a critical component of the drug development process. Confirming that a drug candidate engages its proposed target in the cell and determining the concentration at which it exerts the desired effect(s) fulfill fundamental criteria for translation to activity and efficacy in its target tissue. Thermal shift assays (TSA) are regularly used by industry and academia to uncover or confirm interactions using purified proteins. Recently, this type of assay has been adapted to a cellular format and is called the cellular thermal shift assay (CETSA). In this application note, quantitative and reproducible CETSA data generated with ProteinSimple’s Wes instrument (CETSA-Wes) are presented. This assay verifies drug target binding, and given its quantitative nature, the half maximal inhibitory concentration (IC₅₀) is also calculated.
		Using the Simple Western Total Protein Assay to Normalize Immunoassay Data in the Same Run	[概要表示]
		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.
		Accelerated Serum Biomarker Verification and Validation with Wes and Ella	[概要表示]
		In this application note, we’re honing in on the biomarker verification and validation steps. Proof-of-concept data was generated to demonstrate how Simple Western and Simple Plex assays data give similar trends and work together to give you fast, sensitive, and precise information about your biomarkers of interest.
		Multiplexing with Simple Western

		Simple Western Streamlines Serum Antibody Analysis	[概要表示]
		Scientists measure serum antibody levels to confirm immune responses against a bacteria to diagnose infections, to test for antibody production after vaccination, and to detect the presence of autoantibodies in autoimmune diseases. Traditional Western blots are often used to detect these antibodies, but testing with Western blots means a lot of hands-on time. After the antigen of interest is separated by SDS-PAGE and transferred to a membrane, each lane has to be cut into individual strips so patient serum samples can be individually tested for the presence of specific antibodies. Then you have to process and analyze them manually. Simple Western assays happen in individual capillaries, and everything from sample separation to data analysis is completely automated. No more cutting individual strips, washing and incubating them, or lining them all up with a molecular weight marker before detection. Just pipette your sample into the wells of your assay plate, set up your run, and you're done! And all that manual data analysis is gone too — Compass for Simple Western does it all for you. Did we mention you only need 10 µL of diluted serum per data point? That means you'll get a lot more data points for every 1 µL of neat serum. In this application note, we used Simple Western to detect autoantibodies in lupus patient serum as a model system to generate proof-of-concept data for the assay. But you can use this method any time you need to detect and quantitate specific serum antibodies. In fact, check out how researchers are using this assay to detect Salmonella antibodies without having to cut blots into individual strips.
		Breaking Laser Capture Microdissection Sample Size Road Blocks with Simple Western	[概要表示]
		Laser capture microdissection (LCM) is a powerful tool to identify and isolate a pure sample of the cell type you're interested in. But, proteomic studies with LCM samples are really restricted by the small amounts of tissue collected with each capture since there just isn't much to work with. You often have to use the entire sample captured for traditional Western blot analysis and that only leaves you with one data point! So, researchers often use 2D Electrophoresis and mass spectrometry instead to max out the amount of data they can generate. Both of these methods have their own limitations when it comes to ease-of-use and reproducibility though. Immunohistochemistry is also used to analyze LCM samples as it's a more accessible technique, but it really doesn't give you a lot of info either. Simple Western is an automated capillary-based immunoassay that changes the proteomic research game. You only need 1-10 µL of LCM sample for each data point, so you'll get more data points for each sample you collect. Not to mention the sensitivity that comes with it will even let you analyze proteins you couldn't previously do with traditional Western blot. And it's all wrapped up in a simple workflow that minimizes your hands-on time. Simple Western is a sensitive, easy-to-use analytical tool that ups the ante on protein analysis with LCM samples. In this application note, we'll show you two examples of how Simple Western changed what researchers could do with their precious LCM samples for the better
		High Molecular Weight Protein Analysis Made Simple

		Total Protein Analysis the Simple Western Way	[概要表示]
		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: size- or charge-based Western blotting at the push of a button	[概要表示]
		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.
		Easy Transfer of Your Traditional Western Blot to Wes	[概要表示]
		Wes takes the benefits of Simple Western assays a step further by simplifying their workflow with pre-filled plates and disposable capillary cartridges. This application note gives an overview of how to transfer a traditional Western blot assay to Wes.
		Screening siRNA and Verifying shRNA Knockouts	[概要表示]
		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.
		Detailed Characterization of ERK1 and ERK2 Phosphorylation	[概要表示]
		Monophospho- and diphospho-ERK isoforms are not resolved by traditional Western blot analysis, and the sample quantity required is relatively large. The Firefly ERK1/ERK2 assay can distinguish and quantify unphosphorylated, mono and dual-phosphorylated isoforms of ERK1 and ERK2, allowing a more accurate determination of ERK activation than Western blots provide.