Traditional Western Blot
Primary Antibodies
The success of a Western blot experiment depends on having reliable antibodies validated for Western blot. Bio-Techne's Western blot antibody portfolio includes top-cited monoclonal and recombinant primary antibodies backed by our 100% guarantee.
Featured Western Blot Primary Antibodies
Western Blot Primary Antibodies by Species
Secondary Antibodies & Detection
Bio-Techne offers enzyme- and fluorochrome-conjugated secondary antibodies for use in Western blot experiments. Although enzyme-conjugated secondary antibodies, such as HRP conjugates, are often used in conjunction with chemiluminescence detection and allow for the sensitive detection (low picogram) of antigens, fluorescence-based detection methods have improved signal stability and allow for simultaneous detection of multiple proteins. In fluorescence detection a fluorophore-conjugated antibody directly emits a detectable single, whereas in both chemiluminescent or chromogenic detection the process relies on an enzyme-conjugated antibody plus the addition of a substrate or detection reagent.
Common Conjugated Secondary Antibodies for Western Blot
| AP (enzymatic) | Biotin (enzymatic) | HRP (enzymatic) | Allophycocyanin (APC)/Cy7 |
| DyLight 488 | DyLight 550 | DyLight 650 | DyLight 755 |
| FITC | Janelia Fluor 549 | Janelia Fluor 646 | NL493 |
| NL557 | PE/Cy5.5 | PE/Cy7 |
Western Blot Controls
Controls are vital for Western blot analysis to validate the specificity of protein bands and/or to uncover the root cause of any issues. Bio-Techne provides a range of controls to ensure Western blot success.
| Control Lysates | Over-expression Lysates | Tissue Lysates | Blocking Peptides and Proteins |
| Loading Controls |
Other Western Blotting Support Products
In addition to antibodies and lysate controls, Bio-Techne also offers an assortment of other support products to aid Western blot success, including an HRP stabilizer, sub-cellular fractionation kits, protein ladders, and Western blot membranes.
Western Blot Support Products
Simple Western™ 自动化蛋白质印迹系统
众所周知，蛋白质印迹法劳心费力，难以进行。为了克服传统蛋白质印迹法的难题，来自 Bio-Techne 旗下品牌 ProteinSimple 的 Simple Western 提供全自动毛细管蛋白质印迹分析。蛋白质印迹技术中传统进行的步骤如分离、固定、洗涤和检测在台式毛细管电泳仪内部可自动执行完成。
- Simple Western 提供高通量蛋白质印迹分析，仅 3 小时内就获得多达 25 份样本的全定量结果，或过夜获得 96 份样本的全定量结果。
- 借助 Jess™ 上的 Stellar™ 模块，Simple Western 用户获享业界领先的近红外/红外 (NIR/IR) 荧光检测灵敏度。
- Simple Western 用 RePlex™替代传统蛋白质印迹检测中洗膜及重新上抗体检测，去除了第一轮检测的抗体，用新鲜抗体进行第二轮检测或总蛋白归一化。
Jess 上的 Simple Western 对比传统蛋白质印迹
现在，您可用 Milo™ 在单细胞中进行蛋白质印迹分析
对单细胞蛋白质组学感兴趣？ Milo™ 是我公司在单次运行中测量约 1,000 个单细胞中蛋白质表达的 Single-Cell 蛋白质印迹技术平台。借助全球首项 Single-Cell 蛋白质印迹技术 Milo，验证您的 RNAseq 数据、分析流式细胞荧光分选技术 (FACS) 所分选的细胞并深入了解单细胞蛋白质组学。
相关的蛋白质印迹资源
Background Information
蛋白质印迹法是一种使用抗体鉴定细胞或组织裂解物中蛋白质的实验室方法。归因于抗体结合的特异性质，蛋白质印迹分析可用于检测和定量数千种不同蛋白质混合物中的单一蛋白质。在蛋白质印迹法中，将样本加载到聚丙烯酰胺凝胶上，并根据其分子量通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离。使用的聚丙烯酰胺凝胶由两部分组成：成层胶和分离胶。成层胶含有较少丙烯酰胺且具有较低 pH。这种环境允许样本中的蛋白质形成高度限定的鲜明条带。蛋白质从成层胶进入分离胶，后者碱性更强且具有较高凝胶浓度。这导致蛋白质按大小分离，蛋白质越小，穿过凝胶越快。然后通过施加电流将蛋白质转移到膜上。可以将该膜与目的靶蛋白特异性一抗孵育。施加与酶或荧光色素结合的二抗到膜上，以可视化蛋白质/抗体复合物。条带将在其大致分子量处显现，如分子量阶梯所标记。
Overview of Conventional Western Blot Protocol
(A) Proteins are separated by polyacrylamide gel electrophoresis (PAGE) and (B) transferred to a membrane (e.g. nitrocellulose or PVDF) for detection. (C) The membrane is probed with a primary antibody specific for the target protein and typically followed by an enzyme conjugated secondary antibody to detect the antibody-antigen complex. The enzyme (e.g. horseradish peroxidase, HRP) acts on a substrate (e.g. electro-chemiluminescence, ECL) to emit light, (D) generating a signal captured on autoradiography film or a chemiluminescence imaging system. Learn more about perfecting Western blots.
After completing a Western blot, the membrane can be stripped to remove the primary and secondary antibodies, allowing for the membrane to be incubated with additional antibodies. For more information on this process, view our Membrane Stripping and Reprobing protocol.
什么是 SDS-PAGE？
SDS-PAGE（十二烷基硫酸钠聚丙烯酰胺凝胶电泳）是一种常用于根据大小分离蛋白质的电泳方法。SDS 是一种阴离子去垢剂，它带有负电荷，与蛋白质结合并破坏负责蛋白质三维结构的非共价力，从而使蛋白质变性。SDS 还向蛋白质给予与其大小相关的均匀净负电荷。这导致变性的蛋白质在电泳期间仅根据其大小迁移穿过凝胶。研究人员通常在蛋白质印迹前进行 SDS-PAGE。SDS-PAGE 分离蛋白质，而蛋白质印迹涉及将蛋白质从凝胶转移至膜，并利用抗体确认蛋白质的存在/不存在/表达水平。
什么是天然 PAGE 及为何可能使用它？
天然或非变性凝胶不使用 SDS，也不向上样缓冲液添加还原剂，如二硫苏糖醇 (DTT) 或 β-巯基乙醇。这意味着蛋白质维持其天然结构和电荷。因此，这些蛋白质在电泳期间根据其质量和电荷二者迁移穿过凝胶。天然凝胶用于确定蛋白质的聚集状态、研究蛋白复合物和分离酶。
应上样多少样本用于蛋白质印迹法？
蛋白质印迹实验中，上样蛋白质的量取决于若干因素，包括蛋白和被检测蛋白的表达水平。作为经验法则，对于细胞与胞核裂解物和膜样本，每孔加载 20-30 µg 的总蛋白质。如果检测纯化蛋白，通常上样10至 100 ng 蛋白质。但是，应在蛋白质印迹实验开始前，确定合适的蛋白质上样量。
蛋白质印迹需要什么类型的对照？
适当的蛋白质印迹对照对确定问题起源和验证结果重要。蛋白质印迹实验应包括以下几种对照。
1. 阳性对照裂解物 – 阳性对照裂解物来自已知表达目的蛋白的细胞系或组织样本。这种对照会在蛋白质印迹时产生阳性条带。此对照重要，原因是它确保蛋白质印迹实验方案没有问题，并确保任何阴性结果归因于样本中缺乏蛋白质，而不归因于程序问题。
2. 阴性对照裂解物 – 阴性对照裂解物来自已知不表达目的蛋白的样本。这种对照不会在蛋白质印迹时产生条带。此对照对确定抗体非特异性结合重要。
3. 阳性内源性对照裂解物 - 阳性内源性对照裂解物来自已知表达目的靶标的样本。测试重组蛋白样本（如标记的蛋白质）时应使用此对照。重组蛋白的折叠可能异于天然蛋白质，并且错误折叠可能阻止抗体接近表位。此对照将会让研究人员知道，阴性结果归因于表位遭阻断，而不是实验方案不起作用。
4. 上样对照 – 上样对照是管家基因蛋白，它们是在几乎所有组织和细胞中以同等水平表达的蛋白质。此对照确保孔间蛋白表达差异不归因于上样误差或蛋白转移误差。孔间蛋白质水平的半定量分析需要上样对照。
Simple Western 与蛋白质印迹之间有什么差异？
Simple Western 分析是基于毛细管电泳无缝衔接免疫检测的全自动蛋白质印迹系统。该平台使涉及电泳技术和蛋白质印迹技术的所有步骤（如蛋白质上样和分离、免疫探测、洗涤、检测和数据定量分析）自动化。查看 Bio-Techne 的 Simple Western 概述页面，更多了解 Simple Western。
In general, as proteins migrate through a PAGE gel matrix, they are separated according to their size (MW). The smaller the protein, the faster it migrates through the gel. However, migration may also be affected by other factors. Therefore, the actual band size observed may differ from the predicted size. Some of these factors include post-translational modification, post-translational cleavage, splice variation, relative charge variation, and the forming of protein multimers.
The blocking buffer that is often employed in our Western Blot QC validation is either 3% BSA (Catalog # 5217), or 5% NFDM (Non-fat Dry Milk). In certain cases, a mixture of both BSA and NFDM will be employed. Ensure that BSA is used as the blocking buffer for antibodies that target phosphorylated post-translational modifications. This is to ensure that the antibody does not non-specifically bind the casein found in NFDM.
As a general guideline in Western blot to detect any phospho-protein, it is usually recommended to use 5% w/v BSA (in TBST) because milk contains casein which is a phosphoprotein and as a result it can cause high background by detecting the casein present in milk.
View our Western Blot Troubleshooting Guide.