tp钱包官方网下载|wb分离胶浓度的选择

作者： tp钱包官方网下载

2024-03-09 21:52:57

WB各胶的配置 - 知乎

WB各胶的配置 - 知乎切换模式写文章登录/注册WB各胶的配置羽羽科研小白，啥都不懂，还在学习中┉胶浓度分离范围：6%57~212kd8%36~94kd10%20~80kd12%12~60kd15%10~43kd下层7.5ml，上层2.5ml;配制6%的PAGE分离胶：成分所需各成分的体积（ML）凝胶体积51015202530蒸馏水2.65.37.910.613.215.930%丙烯酰胺混合液1.02.03.04.05.06.01.5mol/L Tris（pH8.8）1.32.53.85.06.37.510%SDS0.050.10.150.20.250.310%过硫酸铵0.050.10.150.20.250.3TEMED0.0040.0080.0120.0160.020.024配制8%的PAGE分离胶：成分所需各成分的体积（ML）凝胶体积51015202530蒸馏水2.34.66.99.311.513.930%丙烯酰胺混合液1.32.74.05.36.78.01.5mol/L Tris（pH8.8）1.32.53.85.06.37.510%SDS0.050.10.150.20.250.310%过硫酸铵0.050.10.150.20.250.3TEMED0.0030.0060.0090.0120.0150.018配制10%的PAGE分离胶：成分所需各成分的体积（ML）凝胶体积51015202530蒸馏水1.94.05.97.99.911.930%丙烯酰胺混合液1.73.35.06.78.310.01.5mol/L Tris（pH8.8）1.32.53.85.06.37.510%SDS0.050.10.150.20.250.310%过硫酸铵0.050.10.150.20.250.3TEMED0.0020.0040.0060.0080.010.012配制12%的PAGE分离胶：成分所需各成分的体积（ML）凝胶体积51015202530蒸馏水1.63.34.96.68.29.930%丙烯酰胺混合液2.04.06.08.010.012.01.5mol/L Tris（pH8.8）1.32.53.85.06.37.510%SDS0.050.10.150.20.250.310%过硫酸铵0.050.10.150.20.250.3TEMED0.0020.0040.0060.0080.010.012配制15%的PAGE分离胶：成分所需各成分的体积（ML）凝胶体积51015202530蒸馏水1.12.33.44.65.76.930%丙烯酰胺混合液2.55.07.510.012.515.01.5mol/L Tris（pH8.8）1.32.53.85.06.37.510%SDS0.050.10.150.20.250.310%过硫酸铵0.050.10.150.20.250.3TEMED0.0020.0040.0060.0080.010.012配制PAGE浓缩胶：成分所需各成分的体积（ML）凝胶体积123456810蒸馏水0.681.42.12.73.44.15.56.830%丙烯酰胺混合液0.170.330.50.670.831.01.31.71.5mol/L Tris（pH6.8）0.130.250.380.50.630.751.01.2510%SDS0.010.020.030.040.050.060.080.110%过硫酸铵0.010.020.030.040.050.060.080.1TEMED0.0010.0020.0030.0040.0050.0060.0080.01Western bolt 洗脱液的配方( 4 ℃ 保存)：100mmol/L β-巯基乙醇；2% SDS；62.5mmol/L Tris-HCL (PH 6.8);一、具体配方： 14.4 mol/L β-巯基乙醇700 μL（微升）在通风橱中操作；SDS2 g;0.5mol/L Tris-HCL (PH 6.8)12.5 ml超纯水加至100 ml二、具体操作步骤：1、将用TBST洗过的条带后放入加有洗脱液的试管中;2、水浴50~55℃/30min，10min摇晃一次;3、取出条带，用大量的TBST冲洗；4、再用TBST洗膜，10min/3次；5、重新用牛奶封闭，接着做western bolt；发布于 2022-03-14 11:00wb实验研究生医学赞同 27添加评论分享喜欢收藏申请

WB实验步骤关键点合集| Abcam中文官网

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abcam 整理了 WB 实验中不容忽视的小细节，并将精华内容制作成了 WB 实验check list，方便您对照这张表来检查每个实验步骤的关键点。您可以点击图片下载 Checklist。

WB 实验流程通常包括样本制备、电泳、转膜、封闭、抗体孵育和检测 6 个部分。这 6 个部分是环环相扣，每一步的疏忽都可能导致一无所获。

样本选择和处理蛋白分子参与生命活动的各个过程，其功能复杂多样，有些蛋白可能只在部分组织和细胞表达（图1）。有些蛋白可能需要诱导，才能表达或表达量增加（图2）。有些蛋白可能存在翻译后修饰，导致其蛋白分子量大小可能与预测不符（图2，3）。有些蛋白可能有多个异构体或者容易被降解，导致出现多条条带的现象（图2）。因此，只有足够了解您研究的蛋白特性，您才能在样本的选择和处理上得心应手，美美的开启您的 WB 之旅。图1 MMP9在不同细胞系表达水平不同。Lane 1:LoVo全细胞裂解液；Lane 2:Huh7全细胞裂解液；Lane 3:MCF7全细胞裂解液；Lane 4:HeLa全细胞裂解液；Lane 5:Caco-2全细胞裂解液； Lane 6:A549(血清饥饿诱导过夜)全细胞裂解液；Lane 7:A549(血清饥饿诱导过夜，80nM TPA处理24小时)全细胞裂解液；Lane 8:MDA-MB-231(血清饥饿诱导过夜)全细胞裂解液；Lane 9:MDA-MB-231(血清饥饿诱导过夜后，200nM TPA处理24小时)全细胞裂解液；Lane 10:HepG2全细胞裂解液。图2 在HeLa细胞中HIF-1 alpha蛋白需低氧诱导才表达。Lane 1 : 未处理C6 (rat glial tumor glial cell)全细胞裂解液；Lane 2 : 400 µM CoCl2和200 µM MG-132 (ab141003)处理4小时后的C6全细胞裂解液。预测分子量: 92 kDa；检测到分子量: 110 kDa。图3 SQSTM1 / p62蛋白。Lane 1:人肝脏裂解液；Lane 2:人肾脏裂解液；预测分子量: 47 kDa；检测到分子量: 62 kDa。

样本制备样本制备是 WB 实验的开端，裂解液的选择、样本的破碎方法、温度和变性等都会影响样本制备是否成功。以下是一些样本制备过程中需关注的小细节：添加复合蛋白酶抑制剂以避免靶标蛋白降解。如果涉及蛋白修饰的检测，需添加相应的去修饰酶抑制剂，例如对于磷酸化修饰蛋白添加复合磷酸酶抑制剂，以防止蛋白在提取时被去磷酸化。选择合适的裂解液来富集更多靶标蛋白。超声破碎处理细胞以富集更多靶标蛋白。超声处理富集THP-1细胞中的Histone H3蛋白。Lane 1:超声处理THP1全细胞裂解液；Lane 2:未处理THP-1全细胞裂解液。整个样本制备过程中，保持样本置于冰上。一般来说，蛋 95℃变性5-10min。对于有些多次跨膜蛋白，37℃或者70℃ 10min可能效果更好。通过Bradford分析、Lowry分析或BCA分析测定样本蛋白浓度。

电泳电泳可以将靶标蛋白与其它蛋白分开，更易检测。不同浓度的分离胶对不同分子量的的蛋白分离效果是不同的。蛋白的上样量，也会影响是否可以检测到有效信号或者获得漂亮的图片。设置阳性和阴性对照不仅有助于分析蛋白功能，在WB实验失败时也有助于分析失败的原因。在电泳时需要特别关注以下小细节：根据蛋白大小选择合适的电泳胶浓度，以确保不同蛋白的迁移速率与分离程度最优。具体可参考表1。表1 不同浓度的分离胶选择蛋白的分子量大小分离凝胶丙烯酰胺百分比4–40 kDa20%12–45 kDa15%10–70 kDa12.5%15–100 kDa10%25–200 kDa8%对于分子量较小的靶标蛋白(如分子量<25 kDa），请使用较高浓度的分离胶进行电泳。对于分子量较大的靶标蛋白（如分子量>100 kDa），请使用较低浓度的分离胶进行电泳。至少上样20μg总蛋白进行电泳。建议使用阳性和阴性对照。

转膜转膜是指将蛋白从电泳胶上转移到膜上，方便目标蛋白的检测。转膜缓冲液中SDS和甲醇含量对不同分子量蛋白的转膜影响是不同的。PVDF膜孔径的选择依赖于蛋白分子量大小。转膜结束后，丽春红染色使蛋白可视化，方便判断转膜是否成功。甲醇浓度对转膜效率的影响。HeLa全细胞裂解液中DNA PKcs蛋白检测。Lane 1: 转膜缓冲液中甲醇浓度10%；Lane 2: 转膜缓冲液中甲醇浓度20%。丽春红染色判断转膜是否成功。左图：转膜成功；右图：转膜失败。在转膜时需要特别关注以下小细节：对于分子量较大的靶标蛋白，建议在转膜缓冲液中加入SDS至终浓度为0.1%。对于分子量较大的靶标蛋白，建议使用0.45μm的PVDF膜。对于分子量较小的靶标蛋白，建议使用0.22μm的PVDF膜。对于分子量较大的靶标蛋白，建议转膜缓冲液中使用10%甲醇或更低浓度。对于分子量较小的靶标蛋白，建议转膜缓冲液中使用20%甲醇。PVDF膜激活完成后充分清洗，完全去除膜上残留甲醇。转膜开始前请确认胶与膜之间没有任何气泡。建议转膜完成后使用丽春红染色，确定转膜是否成功（如果选择荧光标记检测，请确保丽春红完全清洗干净）。

封闭封闭是使未有蛋白吸附的区域被封闭，以防止抗体的非特异性结合，从而降低背景，提高检测灵敏度。常用的封闭液有5%脱脂奶粉和5% BSA或特定封闭液。在封闭时需要特别关注以下小细节：没有适用于所有体系的封闭液，请选择合适的封闭液。使用ab32034检测p27 KIP1时，不同封闭液封闭效果不同。Lane 1: 2% BSA；Lane 2: 5% BSA；Lane 3: 5% NFDM/TBST。抗体孵育抗体孵育通常包括一抗孵育和二抗孵育，一抗可特异性地和靶蛋白相互结合；二抗能辨认一抗的恒定区（具有物种特异性），具有放大信号的作用。在抗体孵育时需要特别关注以下小细节：在WB实验过程中，请避免干膜情况。请根据产品说明书选择合适的抗体工作浓度。如果不清楚给定的未纯化抗体样品的浓度，请参考表2。表2 抗体使用浓度参考表组织培养上清腹水全抗血清纯化的抗体WB1/1001/10001/5001g/ml浓度估计1-3mg/ml5-10mg/ml110mg/ml建议使用新鲜抗体，不建议抗体重复利用。抗体孵育时，膜尽量不要裁剪抗体孵育后充分洗涤，去除非特异性结合检测二抗通常带有标签，以用作检测。常见检测手段有化学发光和荧光标记等。在检测时需要特别关注以下小细节：化学发光检测显色底物的选择，现配现用，避光处理显色底物全覆盖在膜上，避免不均荧光检测溴酚蓝也会有产生荧光的倾向，最好让它离目的条带远一些以上就是 abcam 整理的在 WB 实验中需要注意的细节！不管你是做哪个靶点，这些技巧都是可以适用的！担心遗漏这些细节点的小伙伴也不要着急，点击图片下载我们为您准备的 WB 实验 check list，边做实验边检查，相信一定会获得满意的实验结果！

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Western blot蛋白预染Maker及SDS-PAGE凝胶浓度的选择 - 知乎

Western blot蛋白预染Maker及SDS-PAGE凝胶浓度的选择 - 知乎切换模式写文章登录/注册Western blot蛋白预染Maker及SDS-PAGE凝胶浓度的选择科研不好找星球上的笔记本，超级科研蛋白质预染marker的应用非常广泛。在分子生物学研究中，它可以用于检测蛋白质的表达、鉴定蛋白质的分子量和进行蛋白质的半定量分析。常应用于凝胶电泳和蛋白质印记，蛋白质阶梯以即用型形式提供，可直接加载到凝胶或NC膜上。所有预染蛋白Marker的表观分子量都会因缓冲条件的不同而发生变化，所以应根据所用凝胶体系使用相匹配的表观分子量，来更准确地定位您的目标蛋白。根据蛋白Maker说明书中的迁移模式示意图，则可知道Marker在各凝胶体系中的表观分子量。SDS-PAGE凝胶电泳通过将混合的蛋白质样品注入到已凝固的聚丙烯酰胺凝胶中，然后通过电场使蛋白质在凝胶中迁移，从而将其分离出来。 PAGE凝胶的孔隙大小可以调整，在SDS-PAGE中，蛋白质被丙烯酰胺基团偶联的十二烷基硫酸钠(SDS)包裹，使得蛋白质带有负电荷。通过调整凝胶的孔隙大小和运行条件，可以实现不同大小的蛋白质的分离。较小的蛋白质能够快速通过凝胶的孔隙，而较大的蛋白质则迁移速度较慢。因此，通过PAGE凝胶电泳，可以实现对不同蛋白质大小的分离。需要注意的是，当处理较大的蛋白质复合物或聚合物时，PAGE凝胶电泳的效果可能受到限制。在这种情况下，可以考虑使用其他分离技术，如凝胶过滤、凝胶渗透色谱等。不同分子量大小的蛋白质需要用不同浓度的SDS-PAGE凝胶进行电泳分离。Western blot中转膜是一种将PAGE凝胶中分离的蛋白质转移到膜上的技术，以便进一步检测和分析蛋白质。对于新手来说，转膜的过程并不是一帆风顺的，甚至是一个相当痛苦的经历。初次接触这个过程时不会料到会有这么多复杂的步骤和麻烦。要找到可靠的WB转膜条件，需要耗费不少时间和精力。其中会遇到一些技术细节问题，实验中一定一定牢记一条：细节决定成败。在转膜中，师兄准备了一份根据蛋白质分子量的不同大小对应转膜时间的小总结。总而言之，Western blot转膜对于每一位科研工作者都是一个具有挑战性的过程。发布于 2023-12-25 20:30・IP 属地湖北蛋白质MakerSDS-PAGE电泳赞同 2添加评论分享喜欢收藏申请

怎样根据蛋白分子量配分离胶和浓缩胶浓度？？-丁香实验

蛋白分子量配分离胶和浓缩胶浓度？？-丁香实验48小时，有问必答丁香实验，轻松科研已收录 10000+ 实验48小时，有问必答丁香实验，轻松科研已收录 10000+ 实验48小时，有问必答登录提问提问我要登录｜免费注册丁香通首页|实验问答|免疫|怎样根据蛋白分子量配分离胶和浓缩胶浓度？？怎样根据蛋白分子量配分离胶和浓缩胶浓度？？写回答相关实验：免疫印迹（immunoblotting）夜雨阑珊2022-03-24预览写回答分享6 个回答府宅2022-03-24有帮助根据目的蛋白的分子量大小选择合适的凝胶浓度，再按照下面的表格配制：

预览dxy_gwrp7ndq2022-03-24有帮助一般actin以下的可以跑12%胶

红线左右可以用12%或10%跑

几百KD的很大的用6%的胶

小蛋白12%的10%的一般都能跑预览迟C迟2022-03-24有帮助分离胶浓度的选择主要取决于目的蛋白的分子量大小，不同的分离胶浓度对不同的目的蛋白分子量的分辨率不一样，以下表格数值仅供参考预览天一湖医者2022-03-24有帮助目标蛋白分子量越小，mono/bis百分比越高，目标蛋白分子量越大，mono/bis百分比越小预览未来92022-03-24有帮助根据蛋白电泳实验指导的书。不同浓度的胶对应不同范围分子量的蛋白预览whilt-shirt2022-03-24有帮助浓缩胶浓度基本不变，20-100kda的分子量选择10%，30-120选择8%，20以下选择12%，120以上选择6%预览相关问答问WB实验抽提蛋白是用的loading buffer浓度？5 回答 2056 围观2022-04-07问wb内参不齐怎么调上样量3 回答 8051 围观2022-03-22问大分子量蛋白（470kDa）如何快速转膜？6 回答 1746 围观2022-03-14相关方法菌落免疫印迹法2023-05-30蛋白印迹2023-05-25DNA印迹2022-02-10推荐阅读蛋白质浓缩蛋白的浓缩方法蛋白浓缩方法提问48 小时有问必答扫一扫实验小助手关注公众号反馈TOP打开

实验技术|蛋白免疫印迹WB（中） - 知乎

实验技术|蛋白免疫印迹WB（中） - 知乎首发于基础小白进阶切换模式写文章登录/注册实验技术|蛋白免疫印迹WB（中）小酷-华美生物华美生物，让科研变得有温度！科研就像赛跑，你比别人跑的快，就比别人多发几篇SCI~上一期，给朋友们带来了实验技术|蛋白免疫印迹WB（上）咱们书接上回，继续干货奉上~蛋白免疫印迹（WB）是基于抗原抗体的特异性结合作用，以检测复杂样品中的某种蛋白，并对其进行半定量分析的一种方法。主要用于靶标蛋白特异性表达的定性或半定量分析，蛋白与蛋白或蛋白与DNA相互作用的后续分析，以及蛋白修饰的鉴定分析。凝胶电泳4.1 SDS-PAGE凝胶利用丙烯酰胺和甲叉双丙烯酰胺的聚合作用，可以形成一种网状的凝胶结构，具有一定的分子筛作用。蛋白在大量SDS胶束包裹下，形成SDS的电荷效应，蛋白分子本身电荷被覆盖，蛋白在电势作用下，只与所包裹的SDS所带负电荷相关，即反映分子量大小。SDS-PAGE的分辨率与所使用交联剂丙烯酰胺和甲叉双丙烯酰胺的浓度相关。不同浓度的交联剂所形成的网状分子筛孔径不同，形成分子筛效应。分离情况如下：分离胶浓度分离范围（kDa）8%70-20010%25-7012%20-5515%15-45✪ 丙烯酰胺凝胶的成分及作用4.2 电泳液的配制与选择常规SDS-PAGE凝胶电泳对于分离30-250 kDa范围内的蛋白具有较好的分离作用，根据蛋白分子量大小的不同，参照分离情况表选择合适的分离胶浓度。1L电泳液配方如下：使用Glycine-Tris-gel电泳体系时，一般电泳条件为恒压，浓缩胶60-80 V，分离胶100-120 V。电压越小，跑的越慢，分离效果更好。但对于分子量低于30 kDa的小分子蛋白，Glycine-Tris-gel体系分辨率不高，无法达到相应的分离效果。Tricine相对于Glycine具有更好的电子迁移率和解离常数，使得小分子蛋白在浓缩胶内具有更好的浓缩效应，且在分离胶内具有更高的分辨率。所以，对于小分子蛋白推荐使用电泳液配方如下：使用Tricine-Tris-gel电泳体系时，一般条件为恒压，电压不宜过大，60-100V左右。4.3 Protein Marker 与内参选择在实验过程中，根据实验需求需要设置相应对照。◎ 分子量指示剂合适范围的蛋白Marker，可指示蛋白分子量大小，并在一定程度上反映电泳效果和转膜效率。按照不同使用特性，目前所使用的Marker大致可以分为3种：普通非预染Marker、预染Marker、曝光Marker。◎ 阳性对照组织或细胞中已证实有相应蛋白表达的样本即阳性对照品。◎ 内参对照由于管家基因编码表达的蛋白在各种组织和细胞中的表达量相对恒定，故而在对蛋白进行定量检测时，需要将此类蛋白作为内参，以校正蛋白定量和上样的误差。另外，内参蛋白还可以用于监测整套实验体系是否成功。由于研究对象的不同，需要根据实验目的对内参蛋白进行选择：此外，在选择内参蛋白时还应注意以下几点：1. 注意某些特定条件下的内参基因表达可能发生变化，而不适于作为内参对照。比如药物诱导或者外界刺激存在时，导致某些管家基因的表达量不再恒定，而是发生一定变化。需要结合具体的实验方案，并查询相关文献综合考虑。2. 目的蛋白与内参蛋白大小应存在差异，便于检测操作和区分。如果选用的内参蛋白大小与目的蛋白大小相差不大时，可以先进行目的蛋白的显影。随后采用一抗二抗去除液洗去目的抗体，再进行内参抗体的孵育与显影。转膜◎ 转膜方式膜介质的选择将电泳分离后的蛋白从凝胶中转移到固相介质上，通常采用湿转和半干转。两种方法原理相同，只是施加电场的机械装置和用于固定胶与膜的方式有所不同，半干转用浸润缓冲液buffer的多层滤纸替代。两种方法比较如下：在转膜时，高电流作用下，短时间内装置产热非常明显，所以转膜过程应采取相关措施，以保持低温环境。湿转时，可以将装置进行冰浴，有利于散热。半干转时，不宜长时间进行电转，所以推荐大分子蛋白（100 kDa以上）采用湿转。对于小分子蛋白和正常分子蛋白，湿转与半干转效率相差不大。对于高丰度，小块胶，建议干转，提高效率。低丰度，大块胶，建议湿转。◎ 膜介质的选择转膜过程中使用的膜介质，目前使用最多的包括两种，即硝酸纤维素膜（NC膜）和PVDF膜。NC膜的结合能力主要是与其纯度相关，纯度越高，与蛋白结合能力更强。但纯度高的NC膜较脆，容易出现破碎的情况。相对于NC膜，PVDF膜具有更强的蛋白结合能力，且其具有更好的化学耐受性。需要注意的是，PVDF膜使用前，需在甲醇中浸泡（>15 s）以活化膜上的正电基团，并在转膜液中平衡一段时间。另外，PVDF膜与NC膜都具有不同孔径大小。对于小分子蛋白（<20 kDa），建议使用0.22 μm孔径的膜，避免出现转过的现象。正常情况下使用0.45 μm孔径的膜即可。◎ 蛋白分离与转膜条件优化对于小分子蛋白的转膜，可从以下几点进行优化：1. 可以增大交联剂浓度，采用15％丙烯酰胺凝胶进行电泳。但对于15 Kda以下的蛋白，此胶的分辨率较低，建议在浓缩胶和分离胶之间增加10％的间层胶，以增加小分子蛋白分辨率。2. 采用Tris-Tricine电泳体系替代Tris-Glycine缓冲体系，达到更好的浓缩作用与分离效果。注意应用该系统时，电压不宜过大，60-80 V为宜。3. 选取0.22 μm孔径的膜介质4. 缩短转膜时间，可采用干转方式进行转膜。对于大分子蛋白的转膜，可从以下几点进行优化：1. 降低交联剂浓度，采用8%-5%丙烯酰胺凝胶进行电泳，但需注意胶浓度越低，越容易破碎，操作时应小心。2. 转膜时，电流适当调大，转膜时间延长，并避免转膜过程中出现高热，推荐湿转4℃过夜。3. 适当降低转膜缓冲液中的甲醇，可促进胶中SDS分子与蛋白的分离，高浓度的甲醇对蛋白有固定作用，而不利于大分子蛋白泳出，可降低甲醇浓度至10%。◎ 转膜效率监测1. 预染Marker的转膜情况在一定程度上反映了蛋白转移效率。2. 对胶进行丽春红染色，根据染色后的条带查看是否转膜成功。此过程为可逆过程，但不适用于尼龙膜的染色。丽春红染色液的配制：5%（V/V）乙酸，0.1%（W/V）丽春红，ddH2O混匀，置于4℃保存。丽春红染色流程如下：将转膜后的PVDF膜或NC膜浸没在丽春红染色液中5-10min振荡。取出印迹膜，用PBS洗涤3×5 min。观察染色的红色条带，记录转膜情况。再次使用PBS洗涤3×5 min，以去除结合的丽春红，便于后续WB检测。3. 对胶进行考马斯亮蓝染色，此染色过程不可逆，但考马斯亮蓝染色灵敏度较丽春红高。考马斯亮蓝染色液的配制：10%（V/V）冰醋酸，45%（V/V）甲醇，0.25%（W/V），ddH2O混匀。考马斯亮蓝脱色液的配制：25%（V/V）甲醇，8%（V/V）冰醋酸，ddH2O混匀。考马斯亮蓝染色流程如下：将转膜后的PVDF膜或NC膜浸没在考马斯亮蓝染色液中，置于水平摇床上缓慢摇动，室温染色1 h（根据胶大小、厚度和温度调整），至胶染成蓝色。倒出染色液，将胶浸入脱色液中，置于水平摇床上缓慢摇动，室温脱色4 h，至蓝色背景脱去，蛋白条带可见。◎ 转膜缓冲液的配制1L转膜缓冲液配制如下：转膜buffer需避光保存，可重复使用多次，但由于甲醇较易挥发，应及时更换使用新鲜转膜缓冲液。封闭◎ 封闭剂的选择固相介质表面材质不均一，有很多细小孔洞。当蛋白被转移至固相介质以后，通过非共价作用力吸附于固相介质表面。但并不是所有位点都吸附了蛋白，故需要将封闭剂吸附到固相介质上，以避免抗体分子直接吸附到膜上，产生假阳性或者高背景的结果。选择封闭剂的原则是封闭剂应能够封闭膜上所有未结合位点，而不干扰目的蛋白的结合，且不与靶蛋白表位进行结合，不与抗体及其他试剂有交叉反应。常用封闭剂如下：不同封闭剂的比较实验如下：需要特别注意的是，对于磷酸化的蛋白，进行检测时不建议使用脱脂奶粉和酪蛋白作为封闭剂，并使用TBST替代PBST。对于封闭剂的选择需要根据结果情况进行相应调整，对于大部分抗体在用脱脂奶粉时，可以达到好的封闭效果，但有些抗体在使用BSA封闭时，能够更好的降低背景。封闭条件一般为室温振荡封闭1h。孵育一抗按照产品说明，对一抗进行适当稀释，一抗稀释液一般与封闭剂组分相同。另外，建议选择已有验证的一抗。另外，一抗建议4℃孵育过夜，使抗原抗体充分结合。对于一抗稀释比例最好做梯度预实验，以确定最佳稀释比例。此时可采用简单的Dot blot法进行摸索。使用NC膜，在膜上依次点上不同上样量的样本，自然风干。待膜完全吸收样本后，进行封闭。随后，按上样梯度对膜进行裁剪，分别采用不同浓度梯度的一抗孵育，相同二抗孵育。最后ECL发光底物孵育显影。观察显影情况进行抗体稀释比范围的初步判定。孵育二抗◎ 实验操作1. 二抗孵育前，用PBST/TBST洗膜3×10 min，以去除未结合的一抗。2. 对二抗进行适当稀释，室温孵育1h。3. 二抗孵育完成后，用PBST/TBST洗膜3×10 min，以去除未结合的二抗，再进行后续实验。◎ 二抗选择1. 物种来源不建议选择来源于鼠或兔来源的二抗，因其与人的同源性较大，容易发生交叉反应，导致高背景。常使用羊或驴来源二抗，所使用二抗必须与一抗种属来源不同，且需根据一抗种属选择抗该种属的二抗。另外，还应注意所使用单克隆一抗的亚型，选择针对一抗亚型的二抗。2. 纯化方式对于目前抗体纯化方式主要有Protein G/A纯化和抗原亲和纯化。前者可与血清中所有抗体IgG分子发生结合，抗原特异性无区分。后者是通过能够与抗体特异性识别的配体或者受体来进行结合洗脱，可以纯化血清中的特异性抗体成分。所以，亲和纯化二抗可大大减少非特异性结合，提高蛋白的检测特异性。3. 合适的标记物WB中最常使用二抗标记物为酶标二抗，如辣根过氧化物酶HRP和碱性磷酸酯酶AP。HRP特异性强，作用底物较为广泛，经济快速，且稳定。AP虽然更为灵敏，但其背景往往更高，且在实验样本中可能存在的内源性磷酸酯酶会干扰实验结果。另外，使用AP标记二抗时，封闭剂也应谨慎选择，避免磷酸酯酶的干扰。不同厂家二抗（HRP标记）的比较实验如下：显影1. 化学发光显色最为经典的HRP化学发光底物Luminol，在H2O 存在下，与辣根过氧化物酶发生酶促反应，发出荧光，灵敏度高，成像性好，可在胶片上进行显影。2. 底物显色HRP的生色底物有多种，其中最常用的是DAB，其通过与HRR反应生成不溶性棕褐色沉淀而显色，其灵敏度高，但需要现配现用，且具有致癌性，操作时应小心操作。3. 荧光显色通过使用合适的荧光二抗，可实现荧光二抗显影，弥补了化学发光和底物显色的定量缺陷。实验流程基本就到这了~下期常见问题解答专属奉上哟~下期预告~1.蛋白样品准备2.蛋白定量3.上样4.凝胶电泳5.转膜6.封闭7.孵育一抗8.孵育二抗9.显影10.常见问题—未完·待续———华美生物·让科研变得有温度！—— 扫码关注“武汉华美生物”官方微信，后台回复【实验技术WB】免费下载实验手册，精彩内容抢先掌握！发布于 2019-03-20 11:31免疫生物实验科研赞同 1178 条评论分享喜欢收藏申请转载文章被以下专栏收录基础小白进阶一口吃成

Western blot 超全攻略—步骤详解与经验交流（二）

ern blot 超全攻略—步骤详解与经验交流（二）

Western blot 超全攻略—步骤详解与经验交流（二）10 赞同1 评论37 收藏在上部分内容中介绍了Western blot 的详细步骤，这一部分就结合个人经验，对操作过程中的的注意事项，易出现的问题及易犯的错误进行复盘，从细节拿捏，就按照上部分内容的操作流程顺序挨个来了哦o(*^＠^*)o1 总蛋白提取总蛋白提取是 Western blot 的第一步，也是关键步骤，要求尽可能的获得所有蛋白质，应注意以下问题：整个过程应在低温下进行（我都是在冰上操作的），以避免细胞破碎释放出的各种酶类的修饰。在合适的盐浓度下，应保持蛋白质的最大溶解性和可重复性。蛋白裂解液一定要加蛋白酶抑制剂（PI）和磷酸酶抑制剂（PPI），保证蛋白质的完整性。样品建议分装成合适的量（比如分装出 20μL用于蛋白质定量），然后于 -20℃ 或 -80℃ 中长期保存，但要注意不要反复冻融，因为会使蛋白的抗原特性发生改变。 2 蛋白定量有条件一定要测定蛋白浓度，之前也有看到基于细胞计数+调内参的Western，我也尝试了，事实是我们不能保证每次处理条件绝对一样，在特定处理下，内参的表达也会发生改变。如果第一次内参不齐，下次调整上样，到底调整多少，也很难估量，做一次western大概需要一天多，第二次内参也没调齐的话就浪费了很多很多时间，远不如在开始时就用蛋白浓度计算得到上样量，这样的上样准确且方便。3 免疫印迹清洗玻璃板：玻璃板用洗洁精轻轻擦洗。两面都擦洗过后用自来水冲，再用蒸馏水冲洗干净后立在筐里晾干（或在烘箱烘干或用吹风机吹干）。玻璃板一定要洗干净，不然到时候胶不匀就可能跑出来波浪形的条带。玻璃板固定时，需对齐放入夹中卡紧，灌胶前可先往玻璃板间灌水进行检漏。配胶：配制分离胶应选择合适的浓度，分子量越大，则胶浓度越小。配制凝胶时要充分混匀，此外应保证试剂的新鲜，特别是过硫酸铵。因为 TEMED 催化过硫酸铵释放相关化学基团，再由过硫酸铵释放的基团催化 Acr/Bis 聚合，所以如果过硫酸铵不新鲜的话加再多的 TEMED 效果也不佳。加入 TEMED需快速摇匀。灌胶：灌胶时掌握好速度，开始可快一些，胶面快到所需高度时要放慢速度。操作时胶一定要沿玻璃板流下，这样胶中才不会有气泡。然后加入无水乙醇（或者ddH2O），液封后的胶凝的更快。加水液封时要很慢，否则胶会被冲变形。上样：加样前样品应先离心，尤其是长时间放置的样品。根据蛋白浓度，计算含 50～100 μg 蛋白的溶液体积即为上样量。当计算得到相同上样质量所需体积后，会发现上样体积是不一样的，保证最后的上样体积和样品中的离子浓度一样也是跑出相同宽度完美条带的必要条件。一般是在蛋白样品中+1×loading补齐至一样浓度，marker也会补相应的loading。上样时，小心不要使样品溢出而污染相临加样孔。上样量一般10-15ul，最好不要超过20μL.电泳：每个实验室电泳时间和电压都有差异。按照各实验室的惯例或者电泳槽的使用说明来操作即可。转膜：转膜这一步真的是很重要了，有好几次我都败给了转膜，以下几点一定要注意：（1）PVDF膜一定要浸泡在甲醇中活化，不活化转不过来；（2）制作转膜三明治一定要按照黑面-海绵-滤纸-电泳胶-PVDF膜-滤纸-海绵-白面的顺序依次放置，放错转不过来；一定要注意赶尽气泡，不然转膜之后有气泡的的地方没转过来，然后影响整个转膜结果。另外，一层一层放置膜和滤纸时，要先把滤纸或者膜的一边放到胶上，然后缓缓把剩下的滤纸覆盖到胶上，可以避免有气泡。孵育抗体：一抗很贵，所以尽量回收。可以用含叠氮钠（起到一定的抑菌作用）的5%BSA进行抗体稀释，4℃放置。一次可以配4-5ml，可以用2-3个月。一抗的最佳工作浓度要自己慢慢摸索，设几个浓度梯度1：500，1：1000，1：2000，慢慢来吧ε=(´ο｀*)))注意上述事项基本上就可以跑出漂亮的条带了。﹉﹉﹉﹉﹉﹉﹉﹉﹉﹉(无情分割)就这么多了，宝子们加油啊！最近刚刚提交了硕士论文，博士也百经波折上岸了，所以有时间就分享分享一些经验，本仙女需要喝个咖啡提提神了，给宝子们安利一款我常喝的口感非常nice的咖啡吧，喜欢的可以收入囊中了，嘿嘿~后期安排一波考博相关的内容吧，比如需要准备什么，注意什么，流程是怎么样的，然后我还收集了很多很多各大top榜学校的生化，制药，药学，食品及公卫相关专业的报考信息及相关老师的联系方式，可以分享给大家，可以节省大量大量的时间呢，有需要的可以直接【➕lqq626646953】，数据有用望请我喝杯小奶茶，备注知乎来的，还有什么要交流补充的记得留言哦！听说点赞收藏的小哥哥，小姐姐们都一夜拿捏了WB，吼吼吼！o(*^＠^*)o编辑于 2022-04-15 · 著作权归作者所有

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Identify, compare, and choose recommended Invitrogen protein gels for SDS-PAGE or native PAGE using the following interactive product selector. Filter and compare selections based on sample type, volume, gel chemistry, separation range, gel size, well format, compatible gel tanks, and other features.How to use this selection guide Learn more about our available protein gel welcome packsSee migration charts

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电泳是根据分子的大小和电荷对其进行分离和分析。我们的电泳实验方案包括制备PAGE 凝胶和内参对照。打印本实验方案。电泳可以是一维的（即一个方向）或二维的。一维电泳用于大多数常规蛋白质或核酸的分离。而二维蛋白电泳用于指纹鉴定（fingerprinting），经过实验设计与优化，可以高度准确地解析细胞中的所有蛋白。下面我们将介绍一维电泳技术。对于二维电泳实验方案，我们推荐参考《Gel Electrophoresis of Proteins: A Practical Approach》（Hames BD 和 Rickwood D 主编，实用方法系列，第 3 版。牛津大学出版社，1998 年）。制备聚丙烯酰胺电泳凝胶(PAGE)阳性对照分子量marker上样与跑胶内参对照搜索经WB应用检测的一抗

制备 PAGE 凝胶聚丙烯酰胺凝胶由两种化合物聚合而成的，丙烯酰胺和 N,N'-亚甲基双丙烯酰胺（简称为 bis）。Bis 是凝胶的交联剂。聚合是通过添加过硫酸铵 (APS) 和 DMAP 或 TEMED 来启动的。这种凝胶是中性、亲水、由亚甲基交联的长链碳氢化合物的三维网状结构。分子在凝胶中的分离是由凝胶内形成的孔径的相对大小决定的。决定凝胶孔径的大小有两个因素：丙烯酰胺总量 (%T) 和交联剂的量 (%C)。当丙烯酰胺的总量增加时，孔径减小。就交联而言，5%C 时孔径最小。%C 的增加或减少，都会增加孔径。凝胶可以购买商业化成品，也可以在实验室中制备（在实验室手册中可以找到配方）。Abcam 实验室使用的是 Optiblot 系列凝胶。不管选择哪种方式，都要仔细选择凝胶的百分比，它决定了蛋白的迁移率和分离度。目标蛋白的分子量越小，丙烯酰胺/bis 的百分比越高。目标蛋白的分子量越大，丙烯酰胺/bis 的百分比越低。参考以下简易指南，根据蛋白的分子量大小选择合适的凝胶百分比。当然，也可以选择使用梯度凝胶。蛋白的分子量大小e凝胶丙烯酰胺百分比4–40 kDa20%12–45 kDa15%10–70 kDa12.5%15–100 kDa10%25–200 kDa8%丙烯酰胺是一种强的累积神经毒素：应始终佩戴手套。按照制造商的说明将凝胶置于电泳槽中，并浸泡在迁移缓冲液中。阳性对照使用阳性对照可以确证实验方案的有效性和正确性，特别是当抗体识别的目标蛋白在样品中不存在时。在设计新实验时，我们强烈推荐使用阳性对照；这会使您对实验方案与结果更有信心。分子量marker分子量 marker 使您能够确定蛋白的分子量大小，并且监控跑胶。市面上有各种分子量marker可供选择。我们提供以下分子量marker：分子量marker (ab48854)Prism 蛋白 Ladder (10-175 kDa) (ab115832)Prism Ultra 蛋白 Ladder (10-180 kDa) (ab116027)Prism Ultra 蛋白 Ladder (10-245 kDa) (ab116028)Prism Ultra 蛋白 Ladder (3.5-245 kDa) (ab116029)上样与跑胶使用专用的凝胶上样枪头或微型注射器将样品完整的加入孔中。注意不要接触到孔的底部，否则会产生扭曲的条带。上样量不要过度。如果样品溢入相邻的加样孔中，会影响实验结果。每孔加入 20-40 µg 总蛋白。凝胶应该浸没在含有 SDS 的迁移缓冲液中，非变性凝胶电泳除外。标准的 PAGE 迁移缓冲液（也称作电泳缓冲液）是 1x Tris-甘氨酸：25 mM Tris base190 mM 甘氨酸0.1% SDSpH 约为8.3按照制造商的推荐时间跑胶；不同机器的时间各不相同（根据电压，跑胶 1 小时至过夜不等）。当染料（迁移的最前沿）到达凝胶底部时，关闭电源。此时蛋白将缓慢地从凝胶中洗脱出来，因此凝胶不要保存在溶液中；应立刻进行转膜。内参对照内参对照是用来确认凝胶中加入的样品是否等量，特别是在比较不同样品中蛋白的表达水平时。它们也可以用于检查样品是否从凝胶转移到膜上。甚至在还没上样或转膜时，也可以使用内参对照来定量每条泳道中的总蛋白。如需要发表文章，使用内参对照至关重要。访问我们的内参对照指南。下表包含常用的内参对照：内参对照样品类型分子量注意事项Vinculin全细胞125 kDaCyclophilin全细胞24 kDaGAPDH全细胞35 kDa一些生理因素（如缺氧和糖尿病）会增加特定细胞类型中 GAPDH 的表达水平。Cofilin 全细胞细胞核细胞膜细胞骨架19 kDaAlpha tubulin全细胞细胞骨架50 kDa由于抗菌剂的抗性，微管蛋白的表达水平可能会有所不同 (Sangrajang S et al., 1998; Prasad V et al., 2000)。Beta tubulin全细胞细胞骨架50 kDa由于抗菌剂的抗性，微管蛋白的表达水平可能会有所不同(Sangrajang S et al., 1998; Prasad V et al., 2000)。Actin全细胞细胞骨架42 kDaBeta actin全细胞细胞骨架40 kDa不适用于骨骼肌样品。细胞生长条件的改变以及与细胞外基质成分的相互作用可能会改变肌动蛋白的合成 (Farmer et al., 1983)。VDAC1/Porin线粒体30 kDaCOX IV线粒体20 kDa许多蛋白的分子量与 COX IV 相近。HSP60线粒体细胞膜60 kDaLamin B1细胞核66 kDa不适用于去除了核膜的样品。HDAC1细胞核55 kDaYY1细胞核45 kDaTBP细胞核35 kDa不适用于去除了 DNA 的样品。PCNA细胞核30 kDaCdk4细胞核细胞膜34 kDaNa-K ATPase细胞膜110 kDaTransferrin血清75 kDa

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Western Blot Sample Preparation Protocol

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›A western blot experiment, or western blotting, is a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Here, you can find a collection of western blot recipes for commonly used protein electrophoresis and western blot buffers and stock solutions, and general western blotting protocols for chemiluminescent and fluorescent detection to guide you through your experiment.If you find this doesn’t work for your specific protein of interest, try our BlotBuilder Product Selection Tool to get a set of recommended products with a personalized western blot protocol.Recipes for Western Blot Buffers and Stock SolutionsWestern Blot Sample Preparation ProtocolChemiluminescent DetectionFluorescent DetectionBuffer RecipesDocuments

Western blot protocol for chemiluminescent detection

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this

chemiluminescent protocol.

Materials

Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 88518, or equivalent)Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)Blocking buffer (e.g. StartingBlock Blocking Buffer, Cat. No. 37543)Incubation trays and containersPrimary antibodies (e.g. Invitrogen western blot validated primary antibodies)Secondary antibodies (e.g. Invitrogen western blot validated HRP antibodies)Chemiluminescent HRP substrate (e.g. SuperSignal West Pico PLUS or SuperSignal West Atto Ultimate Sensitivity Substrate, Cat. No. 34580 or A38555)

Protocol

Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.

PVDF: pre-wet in methanol or ethanol (100%) for 30 seconds, briefly rinse in deionized water, and equilibrate in transfer buffer for 5 minutes.Nitrocellulose: equilibrate directly in transfer buffer for 5 minutes.Follow manufacture instructions for wet, semi-dry, or dry transfer.After protein transfer, wash the membrane in deionized water 4 times for 5 minutes each with agitation to remove all transfer buffer.Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation.Dilute the primary antibody per supplier recommendations in the blocking buffer. Use the antibody dilution calculator to assist with calculating antibody volumes based on incubation volume.Protocol tipsFor wet transfer—Invitrogen Mini Blot Module instructionsFor semi-dry transfer—Invitrogen Power Blotter instructionsFor dry transfer—Invitrogen iBlot 2 transfer device instructionsRecommended primary antibody dilutions to use with Thermo Scientific chemiluminescent substrates. Pierce ECLSuperSignal West Pico PlusSuperSignal West DuraSuperSignal West FemtoSuperSignal West AttoRecommended primary antibody dilutions1:1,000 (0.2–10 µg/mL)1:1,000 (0.2–1.0 µg/mL)1:5,000 (0.02–1.0 µg/mL)1:5,000 (0.01–0.2 µg/mL)1:5,000 (0.2–1.0 µg/mL)Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Ensure the volume of the antibody solution is enough to fully cover the membrane.Wash the membrane 3 times with agitation for 10 minutes each in wash buffer.Prepare dilutions of the conjugated secondary antibody in appropriate volume of wash buffer or alternatively in blocking buffer. Use the antibody dilution calculator to assist with calculating antibody volumes based on incubation volume.Protocol tips At Step 8, if using an enzyme-conjugated primary antibody, proceed to Step 13.

Recommended secondary antibody dilutions to use with Thermo Scientific chemiluminescent substrates. Pierce ECLSuperSignal West Pico PlusSuperSignal West DuraSuperSignal West FemtoSuperSignal West AttoRecommended secondary antibody dilutions1:1,000 - 1:15,000 (0.07–1.0 µg/mL)1:20,000 - 1:100,000 (10–50 ng/mL)1:50,000 - 1:250,000 (4–20 ng/mL)1:100,000 - 1:500,000 (2–10 ng/mL)1:100,000 - 1:250,000 (4–10 ng/mL)Incubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane.Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.Prepare working solution of chemiluminescent substrate based upon manufacture instruction. Suggested volume of ~8–10 mL for mini blots and 15 mL for midi blots (0.1 mL working solution per cm2 of membrane).Incubate the blot with the working solution for 1 min. when using standard ECL substrates or 5 min. when using high-performance substrates, such as SuperSignal substrates.Remove the blot from working solution and drain excess reagent.Place the blot in clear plastic wrap or sheet protector and remove bubbles by rolling with blot roller or glass pipette.Image the blot using film or appropriate imaging system.

Fluorescent western blotting protocol

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this

fluorescent protocol.

Materials

Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 22860, or equivalent)Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)Filtered blocking buffer (e.g. Blocker FL Fluorescent Blocking Buffer, Cat. No. 37565)Incubation trays and containersPrimary antibodies (e.g. Invitrogen western blot validated primary antibodies)Secondary antibodies (e.g. Invitrogen fluorescently labeled highly cross-absorbed secondary antibodies)

Protocol

Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.

1:5,000: 3 µL of secondary antibody in 15 mL wash buffer1:10,000: 1.5 µL of secondary antibody in 15 mL wash buffer1:20,000: 0.75 µL of secondary antibody in 15 mL wash bufferIncubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane and protect the membrane from bright light to prevent photobleaching of the fluorescent dyes.Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.Blots can be imaged immediately while still wet, or alternatively may be dried prior to imaging. Place each blot in a sheet protector or on a clean surface prior to imaging to prevent contamination.Image the blot using an appropriate imaging system with fluorescence detection mode.Protocol tipsFor wet transfer—Invitrogen Mini Blot Module instructionsFor semi-dry transfer—Invitrogen Power Blotter instructionsFor dry transfer—Invitrogen iBlot 2 transfer device instructionsProtocol tips Do not add detergent to blocking buffer, as this may increase background fluorescence.

For typical incubation trays, use at least 15 mL for mini blots and 30 mL for midi blots to fully cover the membrane. Avoid low volumes, as differences in agitation and coverage can produce high or uneven background.Protocol tips The final wash time may be reduced by filling and decanting the tray with distilled water 4 times, then moving forward with three 5-minute washes in wash buffer.

Protocol tips To dry the membrane, place it between two sheets of western blot filter paper to protect it from light exposure while drying. Drying the membrane allows for extended storage of the blot and can reduce exposure times. Store blots in the dark to prevent photobleaching.

Western Blot Buffer Recipes

Stock solutions1 M Tris-HCl, pH 7.6 (100 mL)0.5 M Tris-HCl, pH 6.8 (100 mL)10% SDS (10 mL)1.0% Bromophenol Blue (10 mL)10X Tris Buffered Saline (TBS)10X Phosphate Buffered Saline (PBS)Sample preparation buffersRIPA buffer2X Tris-Glycine SDS Sample buffer (Laemmli buffer)4X LDS Sample BufferElectrophoresis running buffers10X Tris-Glycine SDS Running Buffer10X Tris-Glycine Native Running Buffer20X MOPS SDS Running Buffer20X MES SDS Running Buffer10X Tricine SDS Running BufferTransfer buffer25X Tris-Glycine Transfer Buffer20X Bis-Tris Transfer BufferWash buffersTris-buffered saline with Tween 20 (TBST)Phosphate buffered saline with Tween 20 (PBST)Blocking and stripping buffers recipes5% Nonfat Milk3% BSAStripping BufferGel casting recipesSureCast ReagentsStandalone Reagents

Stock solutions

1 M Tris-HCl, pH 7.6 (100 mL)Tris Base12.11 gDeionized water80 mLAdjust pH to 7.6 with HClDeionized waterto 100 mL0.5 M Tris-HCl, pH 6.8 (100 mL)Tris Base6.06 gDeionized water60 mLAdjust pH to 6.8 with HClDeionized waterto 100 mL10% SDS (10 mL)SDS1.00 gDeionized waterto 10 mL1.0% Bromophenol Blue (10 mL)Bromophenol blue100 mgDeionized waterto 10 mL10X Tris Buffered Saline (TBS)Tris Base24 gNaCl88 gDeionized water900 mLpH to 7.6 with HClDeionized waterto 1000 mLReady-to-use alternative:Pierce 20X TBS Buffer, 500 mL (Cat. No. 28358)10X Phosphate Buffered Saline (PBS)NaCl80 gKCl2 gNa2HPO414.4 gNaH2PO42.4 gDeionized water900 mLpH to 7.0 with NaOHDeionized waterto 1000 mLReady-to-use alternative:Pierce 20X PBS Buffer, 500 mL (Cat. No. 28348)

Sample preparation buffers

RIPA buffer: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (100 mL)NaCl0.88 gNP-401 gSodium deoxycholate1 g10% SDS1 mL1 M Tris-HCl, pH 7.62.5 mLDeionized waterto 100 mLProtease Inhibitor Tablet (Cat. No. A32965)2 tabletsReady-to-use alternative:Thermo Scientific RIPA Lysis and Extraction Buffer, 100 mL (Cat. No. 89900)SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL)Recipe for 2X buffer stock:0.5 M Tris-HCl pH 6.82.5 mLGlycerol2 mL10% (w/v) SDS4 mL0.1% (w/v) Bromophenol Blue0.5 mLDeionized waterto 10 mLThe buffer is stable for 6 months when stored at 4°C.Ready-to-use alternative:Invitrogen Novex Tris-Glycine SDS Sample Buffer (2X) (Cat. No. LC2676)LDS Sample Buffer: 106 mM Tris HCl, 141 mM Tris Base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5.Recipe for 4X buffer stock:Tris HCl0.666 gTris Base0.682 gLDS0.800 gEDTA0.006 gGlycerol4 gSERVA Blue G250 (1% solution)0.75 mLPhenol Red (1% solution)0.25 mLDeionized waterto 10 mLThe buffer is stable for 6 months when stored at 4°C. Do not use acid or base to adjust pH.Ready-to-use alternative:Invitrogen NuPAGE LDS Sample Buffer (4X) (Cat. No. NP0007)

Electrophoresis running buffers

Tris-Glycine SDS Running Buffer: 25 mM Tris Base, 192 mM Glycine, 0.1% SDS, pH 8.3.Recipe for 10X buffer stock:Tris Base29 gGlycine144 gSDS10 gDeionized waterto 1000 mLReady-to-use alternative:Novex Tris-Glycine SDS Running Buffer (10X), 500 mL (Cat. No. LC2675)Tris-Glycine Native Running Buffer: 25 mM Tris Base, 192 mM Glycine, pH 8.3.Recipe for 10X buffer stock:Tris Base29 gGlycine144 gDeionized waterto 1000 mLReady-to-use alternative:Novex Tris-Glycine Native Running Buffer (10X), 500 mL, 500 mL (Cat. No. LC2672)MOPS SDS Running Buffer: 50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7.Recipe for 20X buffer stock:MOPS104.6 gTris Base60.6 gSDS10 gEDTA3.0 gDeionized waterto 500 mLDo not use acid or base to adjust pH.Ready-to-use alternative:NuPAGE MOPS SDS Running Buffer (20X), 500 mL (Cat. No. NP0001)MES SDS Running Buffer: 50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3.Recipe for 20X buffer stock:MES97.6 gTris Base60.6 gSDS10 gEDTA3.0 gDeionized waterto 500 mLDo not use acid or base to adjust pH.Ready-to-use alternative:NuPAGE MES SDS Running Buffer (20X), 500 mL (Cat. No. NP0002)Tricine SDS Running Buffer: 100 mM Tris Base, 100 mM Tricine, 0.1% SDS, pH 8.3.Recipe for 10X buffer stock:Tris Base121 gTricine179 gSDS10 gDeionized waterto 1000 mLThe buffer is stable for 6 months when stored at room temperature. Do not use acid or base to adjust pH.Ready-to-use alternative:Novex Tricine SDS Running Buffer (10X), 500 mL (Cat. No. LC1675)

Transfer buffer recipes

Tris-Glycine Transfer Buffer: 12 mM Tris Base, 96 mM Glycine, pH 8.3.Recipe for 25X buffer stock:Tris Base18.2 gGlycine90 gDeionized waterto 500 mLReady-to-use alternative:Novex Tris-Glycine Transfer Buffer (25X) 500 mL (Cat. No. LC3675)Bis-Tris Transfer Buffer: 25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2.Recipe for 20X buffer stock:Bicine10.2 gBis-Tris (free base)13.1 gEDTA0.75 gDeionized water125 mLThe buffer is stable for 6 months when stored at 4°C. Do not use acid or base to adjust pH.Ready-to-use alternative:NuPAGE Transfer Buffer (20X), 125 mL (Cat. No. NP0006)

Wash buffers recipes

Tris-buffered saline with Tween 20 (TBST)10X TBS100 mLTween 201 mLDeionized waterto 1000 mLReady-to-use alternative:Pierce 20X TBS Tween 20 Buffer, 500 mL (Cat. No. 28360)Phosphate buffered saline with Tween 20 (PBST)10X TBS100 mLTween 201 mLDeionized waterto 1000 mLReady-to-use alternative:Pierce 20X PBS Tween 20 Buffer, 500 mL (Cat. No. 28352)

Blocking and stripping buffers recipes

5% nonfat milkNonfat dry milk2.5 gTBST or PBSTUp to 50 mLFilter to remove particulates Ready-to-use alternative:Pierce Clear Milk Blocking Buffer 10X, 100 mL (Cat. No. 37587)3% BSABSA1.5 gTBST or PBSTUp to 50 mLFilter to remove particulates Ready-to-use alternatives:Pierce Blocker BSA (10X) in TBS, 125 mL (Cat. No. 37520)Pierce Blocker BSA (10X) in PBS (Cat. No. 37525)Stripping buffer0.5 M Tris HCl, pH 6.812.5 mL10% SDS20 mL2-mercaptoethanol0.8 mLDeionized water67.5 mLReady-to-use alternatives:Restore Western Blot Stripping Buffer, 500 mL (Cat. No. 21095)Restore Fluorescent Western Blot Stripping Buffer, 100 mL (Cat. No. 62300)

Gel casting recipes

Recipes with SureCast reagentsThe volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. Polyacrylamide %Solution4%6%8%10%12%14%16%18%20%SureCast Acrylamide (40%)0.8 mL1.2 mL1.6 mL2.0 mL2.4 mL2.8 mL3.3 mL3.6 mL4.0 mLSureCast Resolving Buffer2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mL2.0 mLDistilled water5.1 mL4.7 mL4.3 mL3.9 mL3.5 mL3.1 mL2.7 mL2.3 mL1.9 mL10% SureCast APS80 µL80 µL80 µL80 µL80 µL80 µL80 µL80 µL80 µLSureCast TEMED*8 µL8 µL8 µL8 µL8 µL8 µL8 µL8 µL8 µL*Add this last and mix well just before the gel is to be pouredPrepare stacking gel solution according to the following table. The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. Note: Solutions do not require degassing.Solution4%SureCast Acrylamide (40%)0.30 mLSureCast Stacking Buffer0.75 mLDistilled water1.92 mL10% SureCast APS30 µLSureCast TEMED*3 µL*Add this last and mix well just before the gel is to be poured

Recipes with standalone reagents

Stock solutionsPrepare the following stock solutions: all solutions can be stored at room temperature.50% Acrylamide/BIS (29:1)48.3 g Acrylamide1.7 g BIS Bring to 100 mL with water. Store up to two months in a dark glass bottle.Separating Gel Buffer (1 M Tris-HCl, pH 8.8)Add 30.3 g Tris to 150 mL waterAdjust pH 8.8 with HCL Bring to 250 mL with water.Stacking Gel Buffer (0.375M Tris HCl, pH 6.8)Add 11.4 g Tris to about 150 mL waterAdjust to pH 6.8 with HCl Bring to 250 mL with water.Catalyst-Ammonium Persulfate (Make fresh the day of use)100 mg Ammonium Persulfate Bring to 2 mL in water.10% SDS10.0 g SDS Bring to 100 mL with water.50% Sucrose50.0 g Sucrose Bring to 100 mL with water. Separating gelThe following recipes are for approximately 25 mL of separating gel, enough for four 1.0-mm thick mini gels. Scale volumes proportionally based on the number of gels to be cast.Solution6% Gel8% Gel10% Gel12% Gel14% Gel16% Gel18% Gel20% Gel50% Acrylamide/BIS3.0 mL4.0 mL5.0 mL6.0 mL7.0 mL8.0 mL9.0 mL10.0 mLSeparating Gel Buffer9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL9.4 mL10% SDS250 µL250 µL250 µL250 µL250 µL250 µL250 µL250 µL50% Sucrose*4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mL4.0 mLWater7.8 mL6.8 mL5.8 mL4.8 mL3.7 mL2.7 mL1.7 mL750 µLTEMED**6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µL6.25 µLCatalyst**625 µL625 µL625 µL625 µL625 µL625 µL625 µL625 µL*Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. If omitted, increase the amount of water added to make up for the volume of the sucrose solution (increase the water by 4.0 mL for the above tables). **Add these last and mix well just before the gel is to be poured.Stacking gelFollowing recipe is for 4% Stacking Gel (12.5 mL)Solution4%50% Acrylamide/BIS1.0 mLStacking Gel Buffer4.2 mL10% SDS125 µLWater6.3 mLTEMED*5.0 µLCatalyst*1.0 mL*Add these last and mix well just before the gel is to be poured.

Protocols

Chemiluminescent Western Blotting Protocol Fluorescent Western Blotting Protocol Western Blotting Buffer Recipes Personalized Editable Chemiluminescent ProtocolPersonalized Editable Fluorescent Protocol

Technical guides

Chemiluminescence western blotting technical guide and protocolsFluorescent western blotting—a guide to multiplexingFluorescent Western Blotting—an introduction for new users

Chemiluminescent Detection

Western blot protocol for chemiluminescent detection

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this

chemiluminescent protocol.

Materials

Protocol

Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.

Fluorescent Detection

Fluorescent western blotting protocol

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this

fluorescent protocol.

Materials

Protocol

Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.

Buffer Recipes

Western Blot Buffer Recipes

Stock solutions

Sample preparation buffers

Electrophoresis running buffers

Transfer buffer recipes

Wash buffers recipes

Blocking and stripping buffers recipes

Gel casting recipes

Recipes with standalone reagents

Documents

Protocols

Technical guides

Chemiluminescence western blotting technical guide and protocolsFluorescent western blotting—a guide to multiplexingFluorescent Western Blotting—an introduction for new usersEducation center homepageWestern blotting troubleshootingWestern blotting videosFor Research Use Only. Not for use in diagnostic procedures.

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Introduction to Western Blotting

Western Blotting Electrophoresis Techniques

Overview

Sample Prep

Electrophoresis

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Immunodetection

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Image Analysis

Better Electrophoresis

Theory and advice for the best separation.

On This Page

Electrophoresis Basics

Gel Types

Buffer & Gel Systems

Protein Standards

Power Settings

Electrophoresis Tips

Protocols & Resources

Electrophoresis Basics

How Protein Electrophoresis Works

The term electrophoresis refers to the movement of charged molecules in response to an electric field. Electrophoresis has been developed into a standard molecular biology laboratory technique, where molecules of different sizes and charges are transported through a medium at different rates by an applied electrical field, resulting in their separation.

In an electric field, proteins move toward the electrode of opposite charge. Proteins have a wide range of sizes and shapes and have charges imparted to them by their constituent amino acids. As a result, proteins have characteristic migration rates that can be exploited for separation. The total charge on a protein in an aqueous medium depends on the degree of ionization of its acid and base groups, which can be controlled by buffering. Because the mass of a protein is constant, its charge-to-mass ratio is thereby altered. Protein conformation can also be changed by buffering, detergents, and oxidation/reduction. These factors allow for differences in protein migration rates to be tuned to a degree, thus enhancing separation. In protein electrophoresis, the proteins move through and aqueous polymer gel, which retards migration and retains them for imaging and analysis. The rate proteins move in gel electrophoresis is governed by relationships among the characteristics of the electrophoresis system and the proteins.

Factors affecting protein migration include:

Gel composition and strength (weight percentage of polymer)

Electric field strength

Temperature

Protein size, shape, and charge — which are in turn affected by

Buffer pH, ion type, and concentration

Addition of detergents or reducing agents

Polyacrylamide Gel Electrophoresis (PAGE) Overview

Almost all methods for protein separation use an aqueous polyacrylamide gel as a size-selective sieve during separation. This polyacrylamide gel is what lends the name to the technique: polyacrylamide gel electrophoresis, or PAGE. As proteins move through the gel in response to an electric field, the gel’s pore structure allows smaller proteins to travel more rapidly than larger proteins. This difference in migration rate between smaller and larger proteins leads to their physical separation in the gel.

In most PAGE applications, the gel is mounted between two buffer chambers, and the only electrical path between the two buffers is through the gel. Usually, the gel has a vertical orientation, and the gel is cast with a comb that creates wells in which the samples are deposited. Applying an electrical field across the buffer chambers forces the migration of proteins into and through the gel. The movement of the proteins through the gel is normally tracked visually by the addition of a dye to the sample, which can be seen moving further down the gel over time. If the gel is run for too long, the proteins can run off the gel.

Schematic of electrophoretic protein separation in a polyacrylamide gel. MW, molecular weight.

Native PAGE

Native PAGE is a method in which proteins are prepared in nonreducing, nondenaturing sample buffer, and electrophoresis is also performed in the absence of denaturing and reducing agents. Since the native charge-to-mass ratio is preserved, protein mobility is determined by a complex combination of factors. Additionally, since protein-protein interactions are retained during separation, some proteins may also migrate together as multisubunit complexes and move in unpredictable ways. Moreover, because the native charge is preserved, proteins can migrate towards either electrode depending on their charge.

Effect of SDS on proteins in aqueous buffer. SDS helps denature proteins so they adopt a rod-like conformation. SDS also adds an overall negative charge to the protein.

Migration of Proteins and Ions

Denatured proteins are loaded into the wells.

Voltage applied and proteins move into gel. Chloride ions present in gel (leading ions) run faster than SDS-bound proteins and form an ion front. Glycinate ions (trailing ions) flow in from running buffer and form a front behind proteins.

Proteins are stacked between chloride and glycinate ion fronts. Percentage of acrylamide increases and the pore size decreases at interface between stacking and resolving gels. Movement of proteins into the resolving gel is met with increased resistance.

The smaller pore size of the resolving gel separates the proteins. Since the charge-to-mass ratio is equal in all the proteins of the sample, separation is based on molecular weight only.

Individual proteins are separated into bands according to their molecular weights.

SDS-PAGE

This method incorporates the detergent sodium dodecyl sulfate (SDS) into the buffer system and has become the most popular form of protein electrophoresis. When proteins are separated in the presence of SDS and denaturing agents, for example, reducing agents to dissociate intra-protein disulfide bonds, they become fully denatured and dissociate from each other. In addition, SDS binds noncovalently to proteins and imparts several characteristics that can be exploited for protein separation:

Since SDS is negatively charged, it masks the intrinsic charge of the protein and provides an overall negative charge to the proteins

A similar charge-to-mass ratio for all proteins in a mixture, since SDS binds at a consistent rate of 1.4 gram of SDS per gram of protein

A long, rod-Iike shape of the proteins instead of a complex tertiary conformation

As a result, the rate at which SDS-bound protein migrates in a gel depends primarily on its size, enabling molecular weight estimation of the individual proteins as they are resolved into separate bands on the gel.

See Our Electrophoresis Products »

Types of Protein Gels

Resolution of proteins of various sizes on gels with different percentages of polyacrylamide. High-molecular-weight proteins are better resolved with low-percentage acrylamide gels, whereas low-molecular-weight proteins are best resolved with a high percentage of acrylamide. Gradient gels are best for protein samples with a wide range of molecular weights.

Gel Preparation

Polyacrylamide gels are prepared in the laboratory from a solution of acrylamide monomer, which can polymerize into long chains, and bis-acrylamide, which can form cross-links between chains. Polymerization occurs with the addition of an activator, usually the oxidizing agent ammonium persulfate (APS), which provides a source of free radicals to initiate polymerization, typically along with a catalyst to promote radical formation, commonly tetramethylethylenediamine (TEMED).

Gel Percentage Selection

Gel percentage is the main determinant of protein migration rates in PAGE. Polyacrylamide gels are characterized by two parameters: total monomer concentration (%T, in g/100 ml) and weight percentage of cross-Iinker (%C). By varying these two parameters, the pore size of the gel can be optimized to yield the best separation and resolution for the proteins of interest. %T indicates the relative pore size of the resulting polyacrylamide gel; a higher %T refers to a larger polymer-to-water ratio and smaller average pore sizes.

The practical ranges for monomer concentration are stock solutions of 30–40%, with different ratios of acrylamide monomer to cross-linker. The designations 19:1, 29:1, or 37.5:1 on acrylamide/bis solutions represent cross-Iinker ratios of 5%, 3.3%, and 2.7%, respectively, with 37.5:1 being the most commonly used ratio for protein separations.

To see how protein bands of various sizes resolve on gels with different gel percentages and buffer chemistry:

See Our Protein Gel Migration Charts »

Single-Percentage vs. Gradient Gels

Gels can be made with a single, continuous percentage throughout the gel (single-percentage gels), or they can be cast with a gradient of total monomer concentration, %T, through the gel (gradient gels). Gel percentages for single-percentage gels are usually between 7.5% and 20%, and for typical gradient gels are 4–15% and 10–20%. Use protein migration charts and tables to select the gel type that offers optimum resolution of your sample.

Gel Percentage Selection Guide

Molecular Weights of Proteins in Your Sample

SingIe-Percentage or Gradient Gel?

Applications

Narrow range

Single-percentage

Use singIe-percentage gels to separate bands that are close in molecular weight. Since optimum separation occurs in the lower half of the gel, choose a percentage in which your protein of interest migrates to the lower half of the gel; higher percentage for small proteins, lower percentage for large proteins.

Broad range

Gradient

Use gradient gels to separate samples containing a broad range of molecular weights. Gradient gels allow resolution of both high- and low-molecular-weight bands on the same gel.

The larger pore size toward the top of the gel permits resolution of larger molecules, while pore sizes that decrease toward the bottom of the gel restrict excessive separation of small molecules.

Unknown range

Gradient then single-percentage

For new or unknown samples, first try a broad gradient such as 4–20% or 8–16%, for a global evaluation of the sample. Then use an appropriate singIe-percentage gel once a particular size range of proteins has been identified.

To select the best gel percentages and buffer chemistry for your samples:

See Our Gel Selection Guide »

Precast Gels vs. Handcast Gels

Handcast gels must be prepared from acrylamide and bis-acrylamide monomer solutions; the component solutions are prepared, mixed together, and then poured between two glass plates to polymerize. Because acrylamide and bis-acrylamide are neurotoxins, care must be taken to avoid direct contact with the solutions and to clean up any spills, and the acrylamide powders present an inhalation hazard. In addition, the solutions must be completely degassed to avoid the formation of bubbles in the gel. Overall, the hand-casting process is labor intensive, is not as controlled as it is by gel manufacturers and contributes to more irregularities and less reproducibility than precast gels.

Precast Gels

Gel Hand-Casting Equipment

Convenience

Ready to use

Requires preparation and mixing of acrylamide solutions and pouring of gels

Consistency and reproducibility

Gel manufacturers tightly control process and gel quality leading to more consistent results.

Hand pouring of individual gels can lead to inconsistent gel quality that can lead to inconsistent results.

Shelf life

Formulations lasting up to 1 year

4 weeks or less

Cost

Can be 2–3x more than hand-casting.

Low-cost

See Our Protein Gels »

Stain-Free Gels

This is a new category of polyacrylamide gel which behaves like a single percentage or gradient gel but contains a unique trihalo compound. This compound reacts with the tryptophan residues in a UV-induced reaction to produce fluorescence, which can be easily detected by a compatible imager either within gels or on a membrane. Activation of the trihalo compounds in the gel adds 58 Da moieties to available tryptophan residues and is required for protein visualization. Because the compound becomes covalently bound to the protein molecules, they can be imaged repeatedly on the gel or on a membrane after transfer, without additional staining and destaining steps. The sensitivity of the system is comparable to staining with Coomassie (Brilliant) Blue for proteins with a tryptophan content >1.5%; sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%.

Learn More about Stain-Free Gel Technology »

Free eBook

Find out how Stain-Free technology can revolutionize your western blotting

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Free eBook

Find out how Stain-Free technology can revolutionize your western blotting

Request Yours Now

Stain-Free Western Blotting

Stain-Free gel technology allow users to instantaneously visualize protein after electrophoresis, verify that all proteins have been transferred out of the gel, and validate that all of the proteins are on the membrane without the need for additional staining/destaining steps. This potentially saves time wasted on western blots with problems that would not otherwise have been detected until the later stages of blot processing and development.

The Stain-Free Western Blotting Workflow — Faster Results, Better Data

Visualize gel immediately after electrophoresis (5 minutes)

Pre-transfer gel stain-free image to check sample integrity and separation quality.

Verify that all proteins have been transferred out of the gel

Post-transfer gel stain-free image to measure transfer efficiency.

Assess transfer efficiency onto the membrane before detection

Stain-free blot image as loading control.

Learn More about Our Proprietary Stain-Free Western Blotting Workflow »

Electrophoresis Buffer Systems and Gel Chemistries

The pH and ionic composition of the buffer system determine the power requirements and heavily influence the separation characteristics of a polyacrylamide gel. Buffer systems include the buffers used to:

Cast the gel

Prepare the sample (sample buffer)

Fill the electrode reservoirs (running buffer)

Most common PAGE applications utilize discontinuous buffer systems, where two ions differing in electrophoretic mobility form a moving boundary when a voltage is applied. Proteins have an intermediate mobility making them stack, or concentrate, into a narrow zone at the beginning of electrophoresis. As that zone moves through the gel, the sieving effect of the gel matrix causes proteins of different molecular weights to move at different rates. Varying the types of ions used in the buffers changes the separation characteristics and stability of the gel.

Gel and Buffer Chemistries for PAGE

Buffers

Precast (Format) Gels

Gel Type

Selection Criteria

Sample

Running

Mini-PROTEAN

Criterion

Handcast

SDS-PAGE

Tris-HCI, pH 8.6

Easy to prepare, reagents inexpensive and readily available; best choice when switching between precast and handcast gels and need to compare results

Laemmli

Tris/glycine/SDS

•

TGX

Laemmli-like extended shelf -ife gels; best choice when long shelf life is needed (Mini-PROTEAN) and traditional Laemmli separation patterns are desired

Laemmli

Tris/glycine/SDS

•

Bio-Rad FastCast

TGX Stain-Free

Laemmli-like extended shelf-life gels with trihalo compounds for rapid fluorescence (Mini-PROTEAN) detection without staining

Laemmli

Tris/glycine/SDS

•

—

Bis-Tris, pH 6.4

Offer longest shelf life, but reagents may be costly

XT MOPS or XT MES

—

•

—

Tris-acetate, pH 7.0

Best resolution of high-molecular-weight proteins; use for peptide sequencing or mass spectrometry applications

XT Tricine or Tris/Tricine/SDS

—

•

Native PAGE

TGX

Laemmli-like extended shelf-life gels; best choice when long shelf life is needed and traditional Laemmli separation patterns are desired

Native

Tris/glycine

•

—

Stain-Free

Laemmli-like gels with trihalo compounds for rapid fluorescence detection without staining

Native

Tris/glycine

—

•

—

TGX Stain-Free

Laemmli-like extended shelf-life gels with trihalo compounds for rapid fluorescence detection without staining

Native

Tris/glycine

•

—

Tris-acetate, pH 7.0

Offer best separation of high-molecular-weight proteins and protein complexes

Native

Tris/glycine

—

•

Peptide Analysis

Tris-Tricine

Optimized for separating peptide and proteins with molecular weight <1,000

Tricine

Tris/Tricine/SDS

•

Isoelectric Focusing (IEF)

IEF

Cast with Bio-Lyte Ampholytes, allow separation by protein pl; contain no denaturing agents, so IEF is performed under native conditions

50% glycerol

IEF cathode and IEF anode buffers

•

—

See Our Protein Gels »

Protein Electrophoresis Standards

Protein standards are mixtures of well-characterized or recombinant proteins that are loaded alongside protein samples in a gel. They are used to monitor separation as well as estimate the size and concentration of the proteins separated in a gel.

Select protein standards that offer:

Good resolution of the proteins in the size range of interest

Compatibility with downstream analysis (for example, blotting)

Protein standards are available as prestained or unstained sets of purified natural proteins or recombinant proteins. Prestained standards allow easy and direct visualization of their separation during electrophoresis and their subsequent transfer to membranes. Although prestained standards can be used for size estimation, unstained protein standards will provide the most accurate size determinations.

Recombinant Standards

Engineered proteins, tight bands

Evenly spaced

Precise MW estimations, confirmed by mass spectrometry

Affinity tags for detection

Recombinant standards are engineered to display specific attributes such as evenly spaced molecular weights or affinity tags for easy detection. These standards often offer absolute molecular weight accuracy confirmed by mass spectrometry. Because they contain a known amount of protein in each band, they also allow approximation of protein concentration.

Natural Standards

Proteins bind to the dye in varying amounts at different sites producing broader bands

Spacing based on MW of natural protein

Natural standards are blended from naturally occurring proteins. Although prestained natural standards are effective for monitoring gel separation and efficiency, they covalently bind the dye in different amounts and at different locations. This may produce broader bands than are seen with recombinant standards, making them less desirable for molecular weight estimations.

See Our Protein Standards »

Electrophoresis Power Settings

Power supplies that are used for electrophoresis hold one parameter constant (either voltage, current, or power). The resistance, however, does not remain constant during a run: resistance changes as discontinuous buffer ion fronts move through the gel. In SDS-PAGE, resistance increases as the run progresses.

Depending on the buffer and which electrical parameter is held constant, heating of the gel may increase or decrease over the period of the run. Use external cooling during long, unsupervised runs. Temperature-controlled runs yield more uniform and reproducible results.

Constant Voltage

If the voltage is held constant throughout a separation, the current and power (heat) decrease as the resistance increases. This leads to increased run times, which allow the proteins more time to diffuse. But this is offset by the temperature-dependent decrease in diffusion rate as temperature decreases. When running multiple gels in parallel, use of constant voltage is often preferred because a single voltage can be specified that is independent of the number of gels run in parallel.

Constant Current

If the current is held constant during a run, the voltage, power, and consequently the heat of the gel chamber increases during the run. Constant current conditions result in shorter but hotter runs than constant voltage runs. This increase in heat increases the rate of diffusion of the proteins, leading to fuzzier bands unless additional cooling is supplied.

Constant Power

Holding the power constant minimizes the risk of overheating. Many power supplies do not offer this mode of power control and gel manufacturers do not often provide recommended settings for constant power.

See Our Power Supplies »

Electrophoresis Tips

Wash out wells.

Prior to loading sample in the gel, wash out each well with a pipet loaded with running buffer from the upper buffer chamber. This helps wash out any bits of polymerized acrylamide as well as residual acrylamide casting solution. This helps avoid misshapen band morphology.

Avoid excess gel heating.

If a gel runs too hot during electrophoresis, bands may “smile,” where the band pattern curves upward at both sides of the gel. Excessive heating can occur if buffer composition is incorrect, not mixed well, or if power conditions are excessive.

Ensure sample buffer ionic strength is correct.

Both excess salts in samples and (conversely), an ionic strength of the sample that is lower than the gel, can lead to bands appearing skewed or distorted.

Do not overload samples.

It is easier to detect rare protein targets if more sample is loaded on a gel. However, excessive loads can lead to vertical streaking during electrophoresis. With heavy sample loads, reducing electrophoresis voltage by 25% may help to minimize streaking.

Quick Tips:

How to Minimize Band Distortion During Gel Electrophoresis for Western Blotting

Watch the video below to learn how to eliminate smiling and frowning during electrophoresis to get the best gel resolution for your western blots.

Electrophoresis Protocols and Resources

Find the right products for you using the free Western Blot Selector Tool

Start Tool

Find the right products for you using the free Western Blot Selector Tool

Start Tool

Western Blotting Protocol Library

Filter by your laboratory set-up and reagents to get a custom western blotting protocol that best fits your needs.

Better Western Blotting Guide

Tips, Techniques, and Technologies from the Western Blotting Experts at Bio-Rad Laboratories

Protein Electrophoresis Guide

(PDF 8.35 MB)

A guide containing electrophoresis theories and techniques plus tips on troubleshooting common problems using Bio-Rad products.

Little Book of Standards

(PDF 5.12 MB)

Complete reference information for all of Bio-Rad’s protein standards and nucleic acid standards.

Western Blot Doctor Troubleshooting Guide

Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.

Fundamentals of Western Blotting Course #2: Gel Electrophoresis and Transfer

Request your Free Electrophoresis & Western Blotting Layout Post-It

This simple tool allows users to keep track of their Western Blotting experiment from sample preparation to imaging.

Request Yours

Get Your Free Protein Standard Temporary Tattoo

You too can sport a Precision Plus Protein Kaleidoscope standard tatto temporarily.

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Western Blot University

Courses designed to make you a western blotting expert.

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