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WB的无敌战神之路——封闭 - 知乎
WB的无敌战神之路——封闭 - 知乎切换模式写文章登录/注册WB的无敌战神之路——封闭PTMBio景杰生物WB做得好,导师捡到宝。对于刚入实验室的热血青铜宝宝,WB可谓处处都是轰炸区,一个不小心,分分钟落地成盒。为了帮助大家快速get变强秘籍,提升吃鸡率,我们在此重磅推出《WB实验全攻略》系列文章,助力各位早日成为实验室MVP,进阶WB无敌战神。在上一期的分享中,我们介绍了如何湿转 (WB的无敌战神之路——转膜),今天将接着介绍封闭,我们开始吧~封闭的目的:蛋白通过非共价作用力吸附于膜表面,但是膜上很多位置并未吸附蛋白,封闭液中的蛋白成分可以与膜表面的空白位置结合,从而避免一抗的非特异性结合,产生非特异条带或者背景杂乱。01|封闭液的选择正确的封闭液可以促使抗原抗体更好的结合。对于封闭液的选择,可以通过抗体说明书确定有无特殊的封闭方法,也可根据实验结果进行相应调整。小编在这里给大家总结了常见的封闭液以及它们的优缺点。Ⅰ 脱脂奶粉脱脂奶粉成分复杂,含有多种蛋白,封闭全面。大部分情况下,脱脂奶粉是首选的封闭液 (浓度2.5-5% w/v),经济实惠,可以达到较好的封闭效果。但因含少量残留的生物素和碱性磷酸酶 (AP),它不适用于生物素标记和AP标记的抗体系统。脱脂牛奶也不适用于磷酸化蛋白的检测分析,因为奶粉中含有的酪蛋白是一种磷酸化蛋白,会导致抗体的非特异性结合,使条带背景变脏。Ⅱ 牛血清白蛋白 (BSA)脱脂奶粉不能用于磷酸化蛋白的封闭,分析磷酸化蛋白理论上要用BSA (浓度2-5 % w/v)。WB结果显示,相较于脱脂奶粉,BSA更适用于磷酸化蛋白的检测。遥想当年,我还不知道磷酸化蛋白封闭需要用BSA,一个磷酸化蛋白我整整跑了一个月,总是背景高,血的教训哪!虽然BSA成分单一,但普通级别的BSA可能含有IgG或其他血清蛋白,这些蛋白会与哺乳动物抗体 (如抗牛、山羊、绵羊、马等二抗) 产生交叉反应,增加非特异性背景信号,因此建议使用不含IgG的BSA产品。相对于脱脂奶粉,BSA不含生物素,对于生物素标记的抗体系统而言,可能BSA是最好的选择。Ⅲ 血清血清如胎牛血清FBS含有大量的蛋白质,可以用于封闭。但与BSA一样,它含有免疫球蛋白和血清蛋白,这些蛋白会和哺乳动物抗体产生交叉反应,增加非特异性背景信号。而且血清价格更加昂贵,需要的浓度更高 (5-10 % w/v),因此在WB实验中血清一般用的较少,它更多地应用于免疫组化和免疫荧光实验中样本的封闭。Ⅳ 明胶明胶是胶原蛋白的产物,从冷水鱼皮肤中提取出来的明胶即使在低温情况下也不会凝固,通常使用浓度为0.1-5 % (w/v)。与BSA和血清不同,它不含任何血清蛋白,因此不会和哺乳动物抗体产生交叉反应,这大大降低了非特异性背景信号。但鱼胶含有内源生物素,因此不适用于生物素标记的抗体系统。Ⅴ 混合封闭液有研究表明[1],混合型液封闭效果 (脱脂奶粉+BSA+FBS+明胶) “秒杀”前四种封闭液。如图1所示,不难看出,混合型封闭液的封闭效果最好,信号最强 (该实验使用的是HRP标记的抗体系统)。图1 不同的封闭液对高分子量蛋白CFTR表达的影响/Nitrocellulose:NC膜;FBS:胎牛血清;Gelatin:明胶;Blotto:脱脂奶粉;Mixture:混合液Ⅵ 酪蛋白相对于AP标记二抗,HRP标记二抗灵敏度更高,酶促反应更快。绝大多数实验室用的都是HRP标记二抗。如果有实验室用的是AP标记二抗,小编建议用1 % w/v的酪蛋白进行封闭。酪蛋白在中性和碱性条件下带有负电,会与带正点的膜相互作用,可以用于AP标记的抗体检测系统,缺点是溶解性相对较差~02|封闭的流程01、转膜结束前30 min,以TBST为缓冲体系 (配方见文末) 配置封闭液,并利用涡旋仪、摇床等仪器使封闭液充分溶解并混匀;02、转膜结束后,在抗体孵育盒中加入适量封闭液,并用镊子夹住条带,将其完全浸泡在封闭液中,置于摇床上室温摇动孵育1-2 h,也可以4 ℃冰箱孵育过夜。03|问题解析WB的结果不理想有很多原因,下面我们就分析一下封闭可能会导致的问题吧~1. 条带上有黑色斑点奶粉未完全溶解。出现黑点最常见的原因是奶粉没有完全溶解,建议利用涡旋仪、摇床等仪器使奶粉充分溶解并混匀,或溶解后离心取上清液使用。图2 条带上有黑色斑点2. 条带背景高封闭时间过短。封闭的目的是使封闭液中的蛋白成分与膜表面的空白间隙结合,从而避免一抗的非特异性结合。封闭时间过短可能会导致一抗与空白间隙非特异性结合,背景变脏,可以增加封闭时间,或提高封闭液浓度。图3 条带背景高3. 条带信号弱过度封闭。条带信号弱,可以适当降低封闭液浓度,或者更换封闭液,封闭时间如果过长也可以考虑降低,但如果原本时间是1 h,不建议再缩短时间。但封闭过度很少见,条带信号弱可能要优先考虑其他条件,比如样品新鲜度、上样量、一抗二抗浓度等。 图4 条带信号弱04|TBST配方发布于 2023-02-16 11:05・IP 属地浙江科学实验赞同 2814 条评论分享喜欢收藏申请
Western blot中为什么要进行封闭这一步骚操作 - 知乎
Western blot中为什么要进行封闭这一步骚操作 - 知乎切换模式写文章登录/注册Western blot中为什么要进行封闭这一步骚操作科研不好找星球上的笔记本,超级科研在做Western blot实验中,会用到固相载体,在这些固相载体表面有很多孔隙(如筛子般孔洞),通过恒流湿转或半干转,凝胶上覆着的蛋白质被转移到膜上,蛋白以机械填补和吸附的方式结合于膜表面。蛋白印记到有很多微米级孔洞的NC膜,但是蛋白并不是连续的,而有很多空隙,抗体也是蛋白,也会被吸附在空的孔,这样就会有很多非特异性的信号。为防止这些位点与抗体结合引起非特异的染色和背景,一般用惰性蛋白质或非离子去污剂封闭膜上的未结合位点来降低抗体的非特异性结合。封闭剂应该是具有封闭所有未结合位点而不替换膜上的靶蛋白、不结合靶蛋白的表位,也不与抗体或检测试剂有交叉反应的特性。抗原或抗体包被时所用的浓度较低,吸收后固相载体表面尚有未被占据的空隙,封闭就是让大量不相关的蛋白质充填这些空隙,从而排斥在其后的步骤中干扰物质的再吸附。封闭液中的蛋白能够狠牢固的结合在空白位置上,这样抗体蛋白就不会被非特异性的吸附到膜上,而只会跟特异性蛋白结合。Western的灵敏度某种程度上受限于封闭做的好不好。Western blot转膜后的封闭液的选择应该根据实验的具体要求和条件来决定,不同的封闭液可能会对实验结果产生不同的影响。除了封闭液的选择,还有一些注意事项会影响Western blot转膜后的封闭效果。首先,封闭液的温度和时间应该根据实验的具体情况来决定。一般来说,低温封闭液可以减少非特异性抗体的形成,但同时也可能会影响特异性抗体的结合。因此,需要根据实验的目的和要求来选择适当的温度和时间。根据实验的目的和条件,可以选择不同的封闭缓冲液。例如,对于蛋白质表达较低的样品,可以选择含有高浓度封闭试剂的缓冲液,以提高非特异性结合的抑制效果;而对于蛋白质表达较高的样品,可以选择含有低浓度封闭试剂的缓冲液,以避免影响特异性条带的表达。其次,封闭液的pH值也会影响非特异性抗体的形成。一些封闭液的pH值可能不适合某些抗原的保护,因此需要根据实验的具体情况来选择适当的pH值。除了封闭试剂的选择,封闭步骤的时间和温度也是影响实验结果的重要因素。一般来说,封闭时间不宜过长,以避免非特异性信号的干扰;同时,温度也要适宜,以促进抗原抗体的有效结合。此外,封闭试剂的浓度和膜的清洗次数等因素也会对实验结果产生影响,需要进行适当的调整和优化。一般是将在膜封闭液中4℃下脱色摇床孵育1-2小时左右,封闭孵育后,在TBST缓冲液中冲洗膜3次(每次5min)。除了提到的脱脂牛奶、BSA、酪蛋白和封闭凝胶溶液,还有一些其他的封闭液也可以用于Western blot转膜后的封闭操作。例如,一些实验室会使用封闭血清,如山羊血清或兔子血清,作为封闭液,它们可以为膜上的抗原提供更好的保护。此外,一些实验室还会使用特定的封闭剂,如抗氧化剂或抑制剂,来减少非特异性抗体的形成。总之,封闭步骤是Western blot实验中不可或缺的重要环节,需要认真遵守实验操作规范,选择合适的封闭试剂和条件,以确保实验结果的准确性和可靠性。封闭液的质量和纯度也是影响封闭效果的重要因素。如果封闭液的质量和纯度不高,可能会影响非特异性抗体的形成,从而影响实验结果的准确性。发布于 2024-01-25 19:05・IP 属地湖北western转膜封闭赞同 3添加评论分享喜欢收藏申请
蛋白免疫印迹( Western Blot)详细操作步骤 - 知乎
蛋白免疫印迹( Western Blot)详细操作步骤 - 知乎首发于细胞生物切换模式写文章登录/注册蛋白免疫印迹( Western Blot)详细操作步骤岚solo原理:蛋白免疫印迹( Western Blot,WB)是将蛋白样本通过聚丙烯酰胺电泳按分子量大小分离,再转移到杂交膜(blot)上,然后通过一抗/二抗复合物对靶蛋白进行特异性检测的方法。 WB 是进行蛋白质分析最流行和成熟的技术之一。本SOP包括Western Blot 操作方法及常见问题分析,有助于成功完成 WB。一、蛋白样本提取制备1 细胞或组织裂解2 蛋白酶和磷酸酶抑制剂3 蛋白定量4 电泳上样样品的准备二、 电泳1 PAGE 胶的制备2 蛋白分子量 Marker3 阳性对照4 内参对照5 上样与电泳三、 转膜与显色( Western Blot)1 胶中蛋白的检测2 蛋白转膜3 膜上蛋白的检测:丽春红4 膜的封闭5 一抗的孵育6 二抗的孵育7 显色四、常见问题分析与解决方案五、试剂及缓冲液配方一、蛋白样本提取制备蛋白样品制备是 Western Blotting 的第一步,是决定 WB 成败的关键步骤之一。蛋白提取总体原则与注意事项包括:1) 尽可能提取完全或降低样本复杂度使得集中于提取目的蛋白(通过采用不同提取方法或选择不同的试剂盒产品)。2) 保证蛋白处于可以溶解状态(通过裂解液的pH 、盐浓度、 表面活性剂、还原剂等的选择实现)。3) 提取过程防止蛋白降解、聚集、沉淀、修饰等(全程保证在冰盒中低温操作,加入合适的蛋白酶和磷酸酶抑制剂)。4) 尽量去除核酸,多糖,脂类等干扰分子(通过加入核酸酶或采取不同提取策略)。5) 样品分装,长期于-80℃中保存,避免反复冻融。1-1 细胞或组织裂解1-1-1 细胞裂解1. 培养的细胞经预冷的 PBS 漂洗 2 次;2. 吸净PBS,加入预冷的裂解液(使用前裂解液中加入蛋白酶和磷酸酶抑制剂)(0.1 ml /106 cells);结合不同培养板及实际细胞数量级及参照下表加入相应量裂解液: 面积(cm2)细胞量(个)裂解液量(μl)96 孔培养板0.320.4-1.0×10520-5024 孔培养板20.3-0.6×10630-6012 孔培养板4.50.6-1.2×10660-1206 孔培养板9.61.2-2.5×106120-2503.5 cm 培养皿81.0-2.0×106100-2006 cm 培养皿212.5-5.0×106250-50010 cm 培养皿550.7-1.5×107700-150025cm2 培养瓶253.0-6.0×106300-60075cm2 培养瓶751.0-2.0×1071000-20003. 用细胞刮子刮取贴壁细胞,将细胞及裂解液温和地转移至预冷的离心管中;不能用细胞刮子刮取的情况可直接冰上裂解30min后用枪多次吹打至细胞完全裂解;4. 4℃摇动 30 min;5. 4℃离心 12000 rpm,20 min;6. 轻轻吸取上清,转移至新预冷的离心管中置于冰上,即为蛋白样本,弃沉淀。蛋白样本暂时不作处理时可以放入-80℃保存。7. 目的蛋白非细胞外基质(ECM)或对胰酶不敏感时也可以采用胰酶消化法收集细胞(即细胞吸去培养基,PBS 漂洗 2 次,加入胰酶消化至脱落,加入预冷的PBS,移入预冷的EP管,离心收集细胞,加入预冷裂解液(使用前加入蛋白酶和磷酸酶抑制剂),依次按上面4,5,6步骤处理。1-1-2 组织裂解1. 用预冷的工具分离目的组织,尽量置于冰上以防蛋白酶水解;2. 将组织块放在圆底的微量离心管或 EP管中,加入液氮冻结组织于冰上均质研磨,长期可保存于-80°C;3. 每约10 mg组织加入约200 μl 预冷的裂解液(使用前加入蛋白酶和磷酸酶抑制剂),冰浴匀浆后置于4℃摇动2h,裂解液体积与组织样本量有适当比例(最终的蛋白浓度至少达到1 mg/ml,理想的蛋白浓度应为1-5 mg/ml).4. 4℃离心 12000 rpm,20 min,轻轻吸取上清,转移至新预冷的微量离心管中置于冰上,即为蛋白样本,弃沉淀。蛋白样本暂时不作处理时可以放入-80℃保存。1-2蛋白酶抑制剂本实验室使用的蛋白酶抑制剂为PMSF,抑制丝氨酸蛋白酶(如胰蛋白酶,胰凝乳蛋白酶,凝血酶)和巯基蛋白酶(如木瓜蛋白酶)。在水液体溶液中不稳定,30min就会降解一半,应在使用前从贮存液中现用现加于裂解缓冲液中。工作浓度一般用1mM,1:100(V/V)加入贮液(100mM PMSF),样品处理超过1h,补加一次。PMSF剧毒,为了安全和健康,请穿实验服并戴一次性手套操作.PMSF严重损害呼吸道粘膜、眼睛及皮肤,吸入、吞进或通过皮肤吸收后有致命危险。一旦眼睛或皮肤接触了PMSF,应立即用大量水冲洗之。PMSF在水溶液中的活性丧失速率随pH值的升高而加快,且25℃的失活速率高于4℃。pH值为8.0时,20μmmol/l PMSF水溶液的半寿期大约为85min,这表明将PMSF溶液调节为碱性(pH>8.6)并在室温放置数小时后,可安全地予以丢弃。除PMSF外,裂解液中还需加入蛋白酶和磷酸酶抑制剂(做磷酸化蛋白时必须加),推荐购商品化蛋白酶和磷酸酶抑制剂复合试剂盒或 COOKTAIL,或按下表配制:备注:其中Sodium orthovanadate 配制活化方法如下:所有步聚均需在通风橱中进行:1). 用双蒸水配制100 mM 正矾酸钠溶液;2). 用盐酸HCl 调至pH 9.0;3). 煮沸至溶液无色,尽量减少水分挥发;4). 冷却至室温;5). 再调pH 至 9.0;6). 再煮沸至无色;7). 重复上述过程,直至溶液煮沸冷却后达pH 9.0;8). 用水定容至原体积;9). 分装保存于- 20°C. 溶液变黄则弃之不用。1-3 蛋白定量BCA 法以牛血清白蛋白 (BSA) 作为标准曲线。基于双缩脲原理,碱性条件下蛋白质将 Cu2+ 还原成 Cu1+, BCA(Bicinchoninic 酸)螯合 Cu1+ 作为显色剂,产生兰紫色并在 562 nm 有吸收峰,单价 Cu1+ 与蛋白呈剂量相关性,灵敏性很高,试管法可测范围 20-2000 μg/ml,微孔法为 0.5-10μg/ml。不易受一般浓度去污剂的干扰。可耐受螯合剂、略高浓度的还原剂的影响,40min内抗干扰能力强。BCA测定方法如下:1) 标准曲线的绘制:取一块酶标板,按照下表加入试剂:孔号01234567蛋白标准溶液(μL)01234567去离子水(μL)2019181716151413对应蛋白含量(μg)051015202530352) 根据样品数量,按 50 体积 BCA 试剂 A 加 1 体积 BCA 试剂 B(50:1)配制适量 BCA 工作液,充分混匀;3) 各孔加入 200μL BCA 工作液;4) 把酶标板放在振荡器上振荡 30sec,37℃放置 30 min,然后在 562nm 下比色测定。以蛋白含量(μg)为横坐标,吸光值为纵坐标,绘出标准曲线;5) 稀释待测样品至合适浓度,使样品稀释液总体积为 20μL,加入 BCA 工作液 200μL,充分混匀,37℃放置 30 min后,以标准曲线 0 号管做参比,在 562nm 波长下比色,记录吸光值;6) 根据所测样品的吸光值,在标准曲线上即可查得相应的蛋白含量(μg),除以样品稀释液总体积(20μL),乘以样品稀释倍数即为样品实际浓度(单位:μg/μL)。1-4 电泳上样样品的准备1-4-1变性、还原蛋白样本一般的抗体只能识别抗原蛋白中的部份序列结构(表位),因此,为使抗体能够达到结合该表位而需要将蛋白样本进行变性,使之打开折叠的空间结构,蛋白变性一般使用含阳离子变性去污剂如 SDS 的上样 buffer (loading buffer),并于 95-100°C 煮沸5 min,对于多次跨膜蛋白,可以于 70°C 加热 5-10 min,本实验室的 上样 buffer 称为5×SDS凝胶还原型加样缓冲液,上样时与样本1:4混合后变性上样即可。SDS是阴离子去污剂、变性剂。氨基酸侧链与 SDS 充分结合形成带负电荷的蛋白质-SDS 胶束,蛋白质-SDS 胶束所带的负电荷大大超过了蛋白质分子原有的电荷量,消除了不同分子之间原有的电荷差异,SDS与强还原剂一起使蛋白分子氢键、疏水键打开,使蛋白质分子线性化。1-4-2 天然和非还原样本某些抗体识别的表位是非连续氨基酸构成的蛋白三维结构,此种情况则需要进行非变性的 WB,抗体的说明书一般会标注,这种非变性电泳不加 SDS,样本也不需煮沸。某些抗体仅识别蛋白的非还原态,如某些 cysteine 基的氧化态,即loading buffer 和电泳液中不加入β-巯基乙醇和或DTT。data-draft-type="table" data-size="normal" data-row-style="normal">蛋白状态凝胶状态loading buffer电泳缓冲液还原—变性还原和变性有β-巯基乙醇或DTT ,有SDS有 SDS还原—天然还原和非变性有β-巯基乙醇或DTT ,无 SDS无 SDS氧化-变性非还原和变性无β-巯基乙醇或DTT ,有 SDS有 SDS氧化-还原非还原和天然无β-巯基乙醇或DTT ,无 SDS无 SDS注:除说明书特别标注之外,一般情况下,均使用变性和还原电泳二、 电泳SDS-PAGE基本原理:1). SDS-PAGE 是在蛋白质样品中加入 SDS 和含有巯基乙醇的样品处理液,SDS 是一种很强的阴离子表面活性剂,它可以断开分子内和分子间的氢键,破坏蛋白质分子的二级和三级结构。2). 强还原剂巯基乙醇(或二硫苏糖醇,DTT)可以断开二硫键破坏蛋白质的四级结构。使蛋白质分子被解聚成肽链形成单链分子。解聚后的侧链与 SDS 充分结合形成带负电荷的蛋白质-SDS 复合物。3). 蛋白质分子结合 SDS 阴离子后,所带负电荷的量远远超过了它原有的净电荷,从而消除了不同种蛋白质之间所带净电荷的差异。蛋白质的电泳迁移率主要决定于亚基的相对分子质量,而与其所带电荷的性质无关。2-1 PAGE胶的制备聚丙烯酰胺凝胶(polyacrylamide gel)是由单体丙烯酰胺(acrylamide,简称 Acr)和交联剂N,N’-亚甲基双丙烯酰胺(N,N’-methylenebisacylamide,简称 Bis)在催化剂(过硫酸胺或核黄素 AP)和加速剂(四甲基乙二胺 TEMED)作用下聚合交联而成的三维网状结构的凝胶。化学惰性强,具有一定的机械强度和透明度。是良好的电泳介质。聚丙烯酰胺凝胶聚合机理是通过提供氧游离基的催化,使体系发生氧化还原作用来完成的。催化体系主要有化学催化(AP-TEMED)和光化学催化(核黄素-TMTED)体系。PAGE胶分为连续系统和不连续系统两大类。连续系统电泳体系中缓冲液 pH 值与凝胶中的相同。带电颗粒在电场作用下,主要靠电荷和分子筛效应。不连续系统中带电颗粒在电场中泳动不仅有电荷效应、分子筛效应,还具有浓缩效应,因而其分离条带清晰度及分辨率均较前者佳。不连续电泳 作用缓冲液 PH凝胶浓度浓缩胶使蛋白样品浓缩pH6.8 Tris-HCl低,2-5%分离胶使蛋白样品分离pH8.8 Tris-HCl高,根据蛋白大小不连续系统的浓缩效应:凝胶层的不连续性:浓缩胶的孔径大,分离胶的孔径小。在电场的作用下,蛋白质颗粒在大孔胶中遇到的阻力小,移动快。而在小孔胶中遇到的阻力大,移动慢。因此,在两层凝胶的交界处,由于凝胶孔径的不连续性使样品迁移受阻而压缩成很窄的区带。缓冲液离子成分和pH的不连续性:HCl易解离出Cl- ,它在电场中迁移率大,走在最前面,故称为快离子或前导离子。电极缓冲液中的甘氨酸在pH6.8 的缓冲液中解离度很小,仅为 0.1-1%,因而在电场中迁移率很小,称为慢离子或尾随离子。蛋白质均带负电荷,在电场中均移向正极,其有效迁移率介于快慢离 子之间,于是蛋白质就在快慢离子间形成的界面处,被浓缩成极窄的区带。当进入pH8.8的分离胶时,甘氨酸解离度增加,其有效迁移率超过蛋白质,因此氯离子和甘氨酸离子沿着离子界面继续前进。蛋白质分子由于分子量大,被留在后面,然后分离成多个区带。根据SDS聚丙烯酰胺的有效分离范围选择分离胶浓度。SDS-PAGE胶有效分离范围聚丙烯酰胺胶浓度%线性分离范围/kDa557-2127.536-941020-801212-601510-43分离胶的配置:将二块玻璃板叠放整齐,用夹子两边夹好,将这二块玻璃板固定在底座上。插入配套梳子,在梳子下缘划线,指示灌胶位置,拔去梳子。在分离胶的配置烧杯中按配方加料,混匀后利用移液器将分离胶(避免气泡产生)滴入二块玻璃板之间,至液面达到梳子下缘1cm 处。用移液器缓慢加入饱和正丁醇或水,注意不要冲乱胶面。静置,待分离胶聚合后,倒去或用滤纸吸去水层。浓缩胶的配制:在浓缩胶的配置烧杯中按配比加料,混匀后即刻用移液器加浓缩胶(避免气泡产生)覆于二块玻璃板之间的分离胶之上至满,轻轻插入梳子(插入梳子时一边倾斜直至全部插入,防止产生气泡)。静置待其凝结后,即制成凝胶板。凝胶制成后最少需放置2h或最好湿盒过夜后才能使用,保证交联反应完全。2-2 蛋白分子量 Marker预染或非预染各种分子量的蛋白,用于标示电泳中蛋白的大小和示踪。2-3阳性对照目的蛋白或明确表达目的蛋白的组织或细胞的蛋白提取物,用于检验整个实验体系和过程的正确性有效性、特别是一抗的质量和效率。建议使用对照,可查阅文献或抗体说明书选择购买或自提对照样本。2-4 内参对照管家基因编码的、很多组织和细胞中都稳定表达的蛋白,用于检测整个 WB 实验过程及体系是否正常工作,并作为半定量检测目的蛋白表达量的标准对照必须设立。内参名称分子量大小适用范围β-actin43kDa胞浆和全细胞GAPDH30-40 kDa胞浆和全细胞Tubulin55 kDa胞浆和全细胞VCDA1/Porin31 kDa线粒体COXIV16 kDa线粒体Lanin B166 kDa细胞核(不适于去除核膜的样本)TBP38 kDa细胞核(不适于去除DNA的样本)2-5上样电泳蛋白抗原上样量为30 ug。根据样品量选择SDS-PAGE电泳玻璃板间隙厚度,一般0.75mm间隙15孔的上样量 < 15uL/孔,1mm间隙10孔的上样量 < 30ul/孔。上样:将二块玻璃板制成的凝胶板子上的夹子卸去,将凝胶板垂直靠在电泳槽里的电源架上,使凝胶板的凹沿面靠向电源架。通常两块凝胶板共用一个电源架。将凝胶板与电源架按要求固定于电源槽内。按要求加入电泳缓冲液,使分别加入在两块凝胶板中间电源架内的电泳缓冲液与加入在电泳槽中的电泳缓冲液互不相通。轻轻地拔去凝胶板内的梳子。取处理后的样品液,用微量进样器吸取适量样品液,将样品液缓缓加入凝胶板内地凹口部位(样品点入处)。注意不要冲散样品。电泳:用二根导线连接电泳槽与电泳仪,注意红色与黑色电极的插头和插口相配。电泳时上层胶使用低电压恒压电泳,打开电源将将电压调到80v(一般15min左右),而在溴酚蓝进入下层胶时使用高压恒压电泳,将电压调到120v至溴酚蓝到达胶的底端处附近即可停止电泳。三、 转膜与显色3-1胶中蛋白的检测电泳后检测蛋白是否迁移正确与平均,可采用锌染(负染)、胶染(blue silver)或考马斯蓝染色检测,如果凝胶中的蛋白需要进行转膜则需可逆的锌染法,否则可以采用不可逆考马斯亮蓝法染色。锌染法(负染):电泳胶用蒸馏水洗30秒,加入含0.1% SDS 的0.2 M 咪唑溶液摇动染色 10-15 min,再用去离子水洗数秒,加入0.2 M ZnSO4溶液摇动直至在暗背景下蛋白出现透明条带(约1min),移去ZnSO4溶液加入去离子水中止显色;胶置于0.1- 0.25 M Tris/0.25 M EDTA pH 8.0 缓冲液中漂洗脱色两次,再置于电转缓冲液中开始转膜。胶染(blue silver)法:电泳胶用蒸馏水洗数30秒,考马斯亮蓝G-250染液室温染色1h至过夜,保持摇匀,回收染液,倒入数次去离子水摇动至脱去多余的染料,蛋白被染成深蓝色。考马斯亮蓝R250法:电泳胶用蒸馏水洗数30秒,考马斯亮蓝R-250染液室温染色1h至过夜,保持摇匀,回收染液,用蒸馏水洗数分钟,倒入脱色液摇匀至脱去多余的染料,蛋白被染成深蓝色。 考马斯亮蓝快速染色脱色方法: a. 电泳结束后,取胶放入适量考马斯亮蓝染色液中,微波炉加热至接近沸腾或刚刚沸腾,立即停止加热。通常对于胶浓度大于10%的胶比较坚韧,在发生煮沸时不易破损;对于胶浓度小于10%的胶,宜尽量避免煮沸,以免出现 胶碎裂的情况。 b. 随后在染色液温度较高的情况下,在室温摇床上摇动5-10分钟。 c. 回收染色液。d. 加入适量脱色液,确保脱色液可以充分覆盖凝胶。e. 微波炉加热至接近沸腾或刚刚沸腾,立即停止加热。 f. 随后在脱色液温度较高的情况下,在摇床上摇动5-10分钟。此时通常可以观察到比较清楚的蛋白条带。 g. 更换新鲜的脱色液,重复步骤e和步骤f,直至蓝色背景基本上全部被脱去,蛋白条带染色效果达到预期。 h. 完成脱色后,可以把凝胶保存在水中,用于后续的拍照等。保存在水中的凝胶会发生溶涨。如需避免溶涨,可以把胶保存在含20%甘油的水中。长期保存可以制备干胶。3-2 蛋白转膜杂交膜的选择是决定 Western blot 成败的重要环节。应根据杂交方案、被转移蛋白的特性以及分子大小等因素,选择合适材质、孔径和规格的杂交膜。用于 Western blot 的膜主要有两种:硝酸纤维素膜(NC) 和PVDF 膜。膜是蛋白印迹实验的标准固相支持物,在低离子转移缓冲液的环境下,大多数带负电荷的蛋白质会与膜发生疏水作用而高亲和力的结合在一起,但在非离子型的去污剂作用下,结合的蛋白还可以被洗脱下来。根据被转移的蛋白分子量大小,选择不同孔径的膜。因为随着膜孔径的不断减小,膜对低分子量蛋白的结合就越牢固。通常用 0.45μm 和 0.2μm 两种规格的膜。大于20kD 的蛋白可用 0.45μm 的膜,小于20kD 的蛋白就要用 0.2μm 的膜了,如用 0.45μm 的膜就会发生“Blowthrough”的现象。最常用于 Western Blot 的转移膜主要是硝酸纤维素(Nitrocellulose, NC)膜和聚偏二氟乙烯(Polyvinylidene Fluoride, PVDF)膜,此外也有用尼龙膜、DEAE 纤维素膜做蛋白印迹。尼龙膜和 NC 膜的特点相似,主要用于核酸杂交。硝酸纤维素(nitrocellulose, NC)膜:NC 与蛋白质靠疏水作用结合,无需预先活化,对蛋白质的活性影响小;非特异性本底显色浅;价格低廉,使用方便。但结合在 NC 上的小分子蛋白质在洗涤时易丢失; NC膜韧性较差,易损坏。聚偏二氟乙烯(Polyvinylidene fluoride, PVDF)膜:与蛋白质亲和力高,用前需在甲醇中浸泡,以活化膜上的正电基团,使其更容易与带负电荷蛋白结合,非常适合于低分子量蛋白的检测。但 PVDF 膜在使用之前必需用纯甲醇浸泡饱和 1-5 秒钟。膜的选择主要根据:1. 膜与目的蛋白分子的结合能力(也就是单位面积的膜能结合蛋白的载量),以及膜的孔径(也就是拦截蛋白的大小);2. 不影响后续的显色检测(也就是适和用于所选的显色方法,信噪比好);3. 如果后继实验有其他要求,比如要做蛋白测序或者质谱分析,还要根据不同目的来挑选不同的转移膜。蛋白因结合 SDS 而带电荷,在电场下从胶中转至膜上,转膜方式分为半干和湿转两种,半干式转膜速度快,而湿式成功率高并特别适合用于分子量大于100KD 的蛋白。湿式转膜三明治排列为:海绵/滤纸/ 胶/ 膜/滤纸/ 海绵,全部紧密排列,特别是胶/膜之间不能留有气泡,三明治安放的方向确认正确,负极方为带负电的胶里的蛋白,向正极方(膜)电迁移。SDS-PAGE电泳完毕,用刀片或薄板将凝胶板的两块玻璃轻轻撬开,使凝胶倾伏在其中一块玻璃板上。用刀片在凝胶上沿分离胶与浓缩胶的交界处,将分离胶切下,并在分离胶的左上角切掉一小角,以标记样品顺序。然后将胶小心移入转膜缓冲液中。剪下与分离胶同样大小的0.45um的PVDF膜,以甲醇浸泡5s。剪下6张同样大小的滤纸,与PVDF膜,胶同时以转移缓冲液平衡15min。在转膜装置上从负极(黑底)到正极放置海绵垫片、滤纸、胶、膜、滤纸、海绵垫片(由下而上),放置时一定要排除气泡,特别是膜与滤纸、胶与膜、滤纸与胶之间。设置转膜电流为恒流,200mA,时间约需40min(蛋白大小不同所需时间不同,一般1KD约为1min)。转完后,取出膜,在与胶相同的位置小心剪去一角,标示电泳方向及吸附有蛋白的膜面。标准的电转缓冲液为 1X Tris-glycine buffer 不含 SDS,但加入 20%甲醇,如果转膜的蛋白分子量大于80KD,则推荐加入 SDS 使之终浓度为 0.1%。PVDF 膜需要浸泡甲醇中 1-2 min,再孵育于冰冷的电转缓冲液中 5 min,胶也需在冰冷的电转缓冲液中平衡 3-5 min,否则转膜时会导致条带变形。电转移缓冲液中 SDS 与甲醇的平衡、蛋白的大小、胶的浓度都会影响转膜效果,如下调整可以增加转膜效率:a) 大蛋白(大于 100 KD )1) 对于大蛋白而言,其在凝胶电泳分离迁移较慢,而从凝胶转出也非常慢,因此对于这种大分子量蛋白应该用低浓度的凝胶, 8% 或更低,但因低浓度的胶非常易碎,所以操作时需十分小心,2) 大蛋白易在凝胶里形成聚集沉淀,因此,转膜时在电转移缓冲液加入终浓度为 0.1%的 SDS,以避免出现这种情况,甲醇易使SDS 从蛋白上脱失,因此应降低电转移缓冲液中甲醇的浓度至 10%或更低,以防止蛋白沉淀。3) 降低电转移缓冲液中甲醇的比例以促进凝胶的膨胀,易于大蛋白的转出。4) 如果使用硝酸纤维素膜,甲醇是必需的,但如果是 PVDF 膜,甲醇可以不必加入电转移缓冲液中,但转膜前PVDF 需用甲醇活化。5) 选择湿式, 4℃ 转膜过夜,以取代半干式转膜。b) 小蛋白(小于 20 KD)1) SDS 妨碍蛋白与膜的结合,特别是对小蛋白更是如此,因此,对于小分子的蛋白,电转移缓冲液中可以不加 SDS 。2) 保持 20% 的甲醇浓度。注意事项:1. 避免直接接触膜,应使用镊子,手指上的油脂与蛋白会封闭转膜效率并易产生背景污斑2. 排列三明治时,尽量用移液器或 15 ml 试管赶除胶与膜之间的气泡,或将三明治放在装有的培养皿中以防止气泡产生,请戴手套!3. 确认裁剪的膜和滤纸与凝胶尺寸相同,否刚导致电流不能通过膜,从而转膜无效4. 鸡抗体易于与 PVDF 膜和其它尼龙膜结合,导致高背景,请替换成硝酸纤维素膜以降低背景。3-3 膜上蛋白的检测:丽春红染色为检测转膜是否成功,无预染蛋白Marker时可用丽春红染色。染色方法:将膜放入 TBST 洗一次,再置于丽春红染色工作液中,在室温下摇动染色 5 min,大量的水洗膜直至水变清无色蛋白条带清晰,(膜也可以用 TBST 或水重新洗后再进行染色)PVDF 膜需用甲醇再活化后用 TBST洗后进行封闭。3-4 膜的封闭杂交膜上有很多非特异性的蛋白质结合位点,为防止这些位点与抗体结合引起非特异的染色和背景,一般用惰性蛋白质或非离子去污剂封闭膜上的未结合位点来降低抗体的非特异性结合。封闭剂应该封闭所有未结合位点而不替换膜上的靶蛋白、不结合靶蛋白的表位,也不与抗体或检测试剂有交叉反应。最常见的封闭剂是 BSA、脱脂奶粉、酪蛋白、明胶和 Tween-20(0.05 - 0.1%)稀溶液 PBST 或者 TBST。Tween-20 的作用:Tween-20 是一种非离子型去污剂,有复性抗原的作用,可提高特异性的识别能力。在做 westen blot 时,用惰性蛋白质或非离子去污剂封闭膜上的未结合位点可以降低抗体的非特异性结合。Tween 这种非离子型去污剂在乳化蛋白时,不破坏蛋白的结构,可减少对蛋白质之间原有的相互作用的破坏。离子型去污剂如 SDS 则破坏蛋白的结构。传统上有两种封闭液:脱脂奶粉或 BSA。脱脂奶粉不能与生物素化或伴刀豆蛋白标记的抗体一起使用,因为脱脂奶粉含有糖蛋白和生物素。如果封闭剂中含磷酸酶,用磷酸化特异性抗体分析磷酸化蛋白受到影响,因为磷酸酶与印记膜上的磷酸化蛋白接触可使之去磷酸化。检测磷酸化抗体时,不能使用酪蛋白/脱脂奶粉作为封闭剂。某些抗体用 BSA 封闭时因不明原因可能会产生比脱脂奶粉更强的信号,请仔细阅读说明书注明的注意事项和膜的特殊的封闭方法。一般封闭条件为:5% 脱脂奶粉或 BSA 溶液室温或者 37℃缓慢摇荡 1-2 h,特殊情况也可 4℃过夜。根据结果情况调整封闭试剂的浓度和类型。封闭完成后进行洗膜,在方形保鲜盒中加入TBST,将膜放入其中,使TBST没过PVDF膜,在摇床上低速震荡10min洗1次。3-5一抗的孵育孵育 Buffer:按抗体说明书建议的稀释倍数,用封闭液稀释一抗,如果说明书没有建议的稀释倍数,则参照一般推荐的稀释倍数(1:1000-1:2000),一抗浓度过高会导致产生非特异性条带。吸尽封闭液后,立即加入稀释好的一抗,室温或 4℃在摇床上缓慢摇动孵育2 h。孵育时间:一抗的孵育时间可从2h至过夜(一般不超过 18 h)不等,取决于抗体与蛋白的亲和性和蛋白的含量丰度,建议使用较高的抗体稀释倍数和较长的孵育时间来保证特异性结合。孵育温度:尽可能低温孵育,如果在封闭液中孵育一抗过夜,应在 4℃进行否则会产生污染而破坏蛋白(降别是磷酸基团)。孵育一抗时需保持适当的摇动使之均匀覆没膜,防止结合不均匀。孵育完成后吸取一抗,在方形保鲜盒中加入TBST,将膜放入其中,使TBST没过PVDF膜,在摇床上低速震荡10min,重复洗3次。3-6二抗孵育用封闭液稀释二抗至抗体说明书规定浓度,将转有蛋白的PVDF膜浸入装有抗体的方形保鲜盒中,震荡孵育1-2 h。孵育完成后吸取二抗,在方形保鲜盒中加入TBST,将膜放入其中,使TBST没过PVDF膜,在摇床上低速震荡10min,重复洗3-5次。3-7化学发光酶促反应比同位素安全且快速,已经成为 Western Blot 的主流检测方法。酶促反应可以搭配不同的底物从而实现不同的显色方法:化学发光和底物显色,前者灵敏度很高,已经达到皮克级别,甚至还有飞克级别的,灵敏度超过了同位素;而后者由于直接显色而操作简便且成本低。辣根过氧化物酶在H2O2 存在下,氧化化学发光物质鲁米诺(luminol,氨基苯二酰一肼)并发光,在化学增强剂存在下光强度可以增大 1000 倍,通过将印迹放在照相底片上感光就可以检测辣根过氧化物酶的存在。发光液(A液、B液)1:1配置(注意避光),用移液器吸取合适量的发光液至覆盖PVDF膜,ECL发光仪上曝光并采集图像。四、常见问题分析与解决方案五、试剂及缓冲液配方1. 试剂:国药AR:丙烯酰胺(Acr)、甲叉双丙烯酰胺(Bis)、过硫酸铵(AP)、Tris、SDS、甲醇、乙醇、甘氨酸(Gly)、冰乙酸、磷酸、硫酸铵(常温保存)。2. 其它试剂:BSA、甘油、β-巯基乙醇、溴酚兰、Tween20、TEMED、丽春红、考马斯亮蓝G250/R250(常温保存);ECL发光液(4℃保存);二抗、蛋白分子量marker、PMSF(-20℃保存)。3. 耗材:PVDF膜(millipore)、滤纸、枪头。4. 缓冲液:30% Acr/Bis(棕色瓶)、10% 过硫酸铵、1×转膜缓冲液、5%BSA或脱脂奶粉封闭液(4℃保存);1.5M Tris-HCl(pH8.8)、1M Tris-HCl(pH6.8)、10% SDS 、5× SDS凝胶还原型加样缓冲液、10× TBS、10×Tris-Gly电泳缓冲液、10×转膜缓冲液、1× Tris-Gly电泳缓冲液、TBST(常温保存)。5. 缓冲液配制:a) 30% Acr/Bis:丙稀酰胺29.2g,甲叉双丙稀酰胺0.8g,双蒸蒸馏水溶解定容至100ml,过滤备用,4°C棕色瓶保存。b) 1.5M Tris-HCl(pH8.8):Tirs 18.2g,溶于蒸馏水中,定容至100mL。加入盐酸调节pH值至8.8,常温保存。c) 1M Tris-HCl(pH6.8):Tirs 12.1g,溶于蒸馏水中,定容至100ml。加入盐酸调节pH值至6.8,常温保存。d) 10% SDS:SDS 20.0g溶于蒸馏水中,定容至200ml,加热至68°C助溶。常温保存。e) 10% 过硫酸铵(AP):过硫酸铵0.1g,溶于1ml 蒸馏水中,现配现用或分装冷冻备用。4°C保存时最多不超过2w。f) 5×SDS凝胶还原型加样缓冲液:0.25 M Tris-HCl (pH 6.8) 1ml、SDS 0.25g、甘油 0.189g、溴酚兰 25mg,双蒸水定容到5ml,使用前加入β-巯基乙醇0.5ml。g) 10× Tris-Gly电泳缓冲液:Tris 30.2g,甘氨酸188g,SDS 10g,加入双蒸水定容至1L。1× Tris-Gly电泳缓冲液:用100ml量筒取100ml 10× Tris-Gly电泳缓冲液,加入到1000ml容量瓶中,用蒸馏水定容到1000ml。h) 10×转膜缓冲液: Tris 30.3g 、甘氨酸151.1g 、定容至800ml。1×转膜缓冲液:10×转膜缓冲液80ml、甲醇200ml, 加蒸馏水720 ml。i) 10× TBS:Tris 24.2g、氯化钠80g,双蒸水定容至1L,调PH 7.6。j) TBST:用100ml量筒取100ml 10× TBS,加入到1L容量瓶中,用蒸馏水定容到1L,再加入1ml Tween 20混合均匀。k) 封闭液:2g BSA或脱脂奶粉,溶于装有40ml TBST的50ml离心管中,配成5% BSA或脱脂奶粉封闭液,现配现用,短期4°C保存,较长时间则冷冻保存。l) 100mM PMSF:称量0.174g PMSF(针状结晶固体)溶于10ml 无水乙醇,震荡混匀,保存在-20°C。m) 2%的丽春红贮备液: 丽春红2g,三氯乙酸30g,磺基水杨酸30g,加水定容至100ml,过滤,常温保存。丽春红染色工作液:2%的丽春红贮备液 1:10 稀释,即加 9 倍的 ddH2O。n)考马斯亮蓝R250染色液:0.25%考马斯亮蓝R250,40%双蒸水, 10% 冰乙酸,50%甲醇混匀,过滤,常温棕色瓶保存。o)考马斯亮蓝R250染色脱色液:常温保存: 67.5%双蒸水,7.5%冰乙酸, 25%甲醇混匀,常温保存。p)考马斯亮蓝G-250染液: 10%磷酸,10%硫酸铵,0.12%考马斯亮蓝G-250, 20%甲醇,常温棕色瓶保存。配制方法:按就次序依次加入10%磷酸,10%硫酸铵,等完全溶解后加入0.12%考马斯亮蓝G-250,等完全溶解后加入20%甲醇混匀,过滤。 分离胶配方 5%浓缩胶配方文章来自:无锡菩禾生物医药技术有限公司编辑于 2020-08-13 15:26蛋白表达细胞生物学分子生物学赞同 7718 条评论分享喜欢收藏申请转载文章被以下专栏收录细
蛋白质印迹(Western blot)实验方案| Abcam中文官网
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蛋白质印迹(Western blot)实验方案
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Western blot 资源
缓冲液与储备液
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β-肌动蛋白抗体 (ab8227) 内参对照
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Western blot 培训
我们的 western blot 实验方案包含溶液、试剂、检测步骤和有用的链接,可指导您完成整个实验。2020 年 12 月 14 日审核Western blot 是一项通过凝胶电泳按照分子量大小分离蛋白,然后再利用特异性抗体识别这些蛋白的技术。免疫检测通常使用由硝酸纤维素或 PVDF(聚偏二氟乙烯)制成的膜。将凝胶紧贴膜放置,蛋白在电流作用下从凝胶迁移到膜上。然后利用目标靶点特异性抗体对膜做进一步处理,再通过二抗和检测试剂让膜显色。目录溶液与试剂:裂解缓冲液溶液与试剂:电泳、转膜及封闭缓冲液样本裂解样本制备上样和跑胶蛋白转膜抗体染色相关链接网络研讨会记录查看下方的 western blot 实验方案视频。
查看更多实验方案视频,请单击访问我们的方案视频库。>> 打印完整的 western blot 实验方案>> 查看我们的 western blot 实验方案图如需提升 western blot 分析技能,请查看我们的免费 western blot 培训(可点播)。
溶液与试剂:裂解缓冲液这些缓冲液在 4 ℃ 下可保存数周,也可分装后在 -20 ℃ 下保存长达 1 年。NP-40 缓冲液150 mM 氯化钠1.0% NP-40(可用 0.1% Triton X-100 代替)50 mM Tris-HCl,pH 8.0蛋白酶抑制剂RIPA 缓冲液(放射免疫沉淀检测缓冲液)150 mM 氯化钠1% IGEPAL CA-6300.5% 脱氧胆酸钠0.1% SDS(十二烷基硫酸钠)50 mM Tris-HCl,pH 8.0蛋白酶抑制剂Tris-HCl20 mM Tris-HCl(三羟甲基氨基甲烷盐酸盐)蛋白酶抑制剂
溶液与试剂:电泳、转膜及封闭缓冲液
Laemmli 2X缓冲液/上样缓冲液4% SDS10% 2-巯基乙醇20% 甘油0.004% 溴酚蓝0.125 M Tris-HCl测定 pH 值并将 pH 值调整至 6.8电泳缓冲液(Tris-Glycine/SDS)25mM Tris base(三羟甲基氨基甲烷游离碱)190mM 甘氨酸0.1% SDS测定 pH 值并将 pH 值调整至 8.3转膜缓冲液(湿转)25mM Tris base (三羟甲基氨基甲烷游离碱)190mM 甘氨酸20% 甲醇测定 pH 值并将 pH 值调整至 8.3对于大于 80 kDa 的蛋白,建议 SDS 终浓度为 0.1%。转膜缓冲液(半干转)48mM Tris base39mM 甘氨酸20% 甲醇0.04% SDS封闭缓冲液3–5% 牛奶或 BSA(牛血清白蛋白)加入 TBST 缓冲液。充分混合后过滤。不过滤可能会有斑点沉积,这种小暗点会在显色时影响实验结果。
样本裂解细胞培养裂解液的制备将细胞培养皿放置冰上并用冰冷的 PBS 洗涤细胞。吸出 PBS,然后加入冰冷的裂解缓冲液(每 107 个细胞/100 mm 培养皿/150 cm2 烧瓶加 1 mL;每 5x106 个细胞/60 mm 培养皿/75 cm2 烧瓶加 0.5 mL)。用预冷的塑料细胞刮刀将贴壁细胞从培养皿上刮下,然后轻轻将细胞悬液转移到预冷的小离心管中。或者,用胰蛋白酶消化细胞并用 PBS 洗涤细胞,然后将细胞重悬浮于小离心管内的裂解缓冲液中。4℃ 下持续振摇 30 分钟。放入微型离心机,在 4°C 下离心。您可能需要根据细胞类型改变离心力和离心时间;指南给出的参考标准是在 12,000 rpm 转速下离心 20 分钟,但须根据您的实验确定(白细胞所需的离心力很小)。轻轻地从离心机中取出离心管放置在冰上。将上清液吸出转移到放置在冰上预冷的新管中,弃去沉淀。组织裂解液的制备3.1 用干净器械解剖目标组织,最好在冰上,并且越快越好以防蛋白酶降解。将组织放入圆底离心管或 Eppendorf 管中,浸入液氮中“速冻”。样本在 -80°C 储存备用,或放在冰上立即匀浆。对于一块约 5 mg 的组织,向管中迅速加入约 300 μL 裂解液,并用电动匀浆器匀浆,2X 裂解液冲洗刀片两次,每次 200 μL,然后在 4℃ 下(例如将回旋振荡器放入冰箱)持续振摇 2 小时。裂解液的体积必须根据组织总量决定;蛋白提取物不宜过稀释,以免造成蛋白损失,并尽量减少样本体积,以便凝胶上样。最小浓度为 0.1 mg/mL,最佳浓度为 1-5 mg/mL。在微型离心机中 4℃ 下按照 12,000 rpm 的转速离心 20 分钟。轻轻地从离心机中取出离心管放置在冰上。将上清液吸出转移到放置在冰上预冷的新管中,弃去沉淀。样本制备取少量裂解液,用于蛋白质定量分析。测定每种细胞裂解液的蛋白质浓度。确定蛋白质的上样量,并添加等体积的 2X 稀释 Laemmli 样本缓冲液。我们建议使用以下方法对样本进行还原和变性,除非在线抗体数据表显示应使用非还原和非变性条件。对样本进行还原和变性时,将样本缓冲液中的细胞裂解液在 100°C 下煮沸 5 分钟。裂解液可等量分装并在 -20°C 下储存备用。上样和跑胶3.1 将等量的蛋白和分子量标志物上样至 SDS-PAGE 凝胶孔中。细胞裂解液或组织匀浆的总蛋白上样量为 20-30 μg,纯化蛋白的上样量为 10-100 ng。3.2 在 100 V 下跑胶 1-2 小时。时间和电压可能需要优化。我们推荐按照制造商的说明进行操作。建议使用还原型凝胶,除非抗体数据表推荐使用非还原性条件。凝胶百分比取决于目标蛋白的大小:蛋白大小凝胶百分比4–40 kDa20%12–45 kDa15%10-70 kDa12.5%15-100 kDa10%25-100 kDa8%也可以使用梯度凝胶。蛋白从凝胶转移到膜膜可以是硝酸纤维素,也可以是 PVDF。用甲醇活化 PVDF 1 分钟,并在制备转膜层之前用转膜缓冲液冲洗 PVDF。转膜时间和电压可能需要优化。我们推荐按照制造商的说明进行操作。可在封闭步骤之前用丽春红染色法检查蛋白质转膜。转膜层的制备如下:
图 1.制备好的转膜层示例。
抗体染色
用封闭缓冲液在室温下封闭膜 1 小时或在 4°C 下封闭过夜。用适当稀释的一抗在封闭缓冲液中孵育膜。我们建议在 4°C 下过夜孵育;其他条件可以优化。用 TBST 洗涤膜 3 次,每次 5 分钟。用推荐稀释度的偶联二抗在封闭缓冲液中室温孵育膜 1 小时。用 TBST 洗涤膜 3 次,每次 5 分钟。产生信号时,请遵循试剂盒生产商的建议。除去多余的试剂,并用透明塑料膜覆盖膜。利用暗室显影技术采集化学发光图像,或利用常规图像扫描法采集比色检测图像。
相关链接
查看更多 western blot 实验方案查看所有 Abcam 内参对照。示例内参对照:ab8227 beta actin
所有泳道:beta Actin 抗体 - 内参对照 (ab8227),稀释度为 1/5000泳道 1:HeLa 全细胞提取物泳道 2:酵母细胞提取物泳道 3:小鼠脑组织裂解液查看我们可提供的阳性对照裂解液、封闭肽和阳性对照蛋白清单。查看蛋白质印迹中表现出色的 AbExcel 二抗。观看我们简单易懂的实验方案视频。实验方案由 Abcam 根据 Abcam 实验室使用 Abcam 试剂和产品开展的实验“按原样”提供;在其他条件下使用实验方案得出的结果可能会有所不同。网络研讨会记录Western blot 的目的在于按照分子量大小在凝胶上分离蛋白。然后将蛋白转移到膜上,从而使用抗体对蛋白进行检测。在含有还原剂(如 β-巯基乙醇)的样本缓冲液中,95 ℃ 下加热样本 5 到 10 分钟。这样可以让线性化蛋白带上与其大小成正比的负电荷。将凝胶放入电泳槽中并加入缓冲液,确保孔的顶部被缓冲液覆盖。所用凝胶的丙烯酰胺百分比取决于靶蛋白的分子量。将分子量标志物上样至第一泳道,然后将样本上样至相邻的孔中。所有样本均含有等量蛋白。所有样本完成上样后,添加电泳缓冲液,给电泳槽盖上盖子。打开电源,按照制造商的推荐设置凝胶槽中凝胶的电压。这时应该能够看到凝胶槽中有上升的气泡。跑胶,直到染料前沿充分移动至凝胶。下一阶段是将蛋白从凝胶转移到膜。膜通常由硝化纤维或 PVDF 制成。从凝胶槽中取出凝胶,并小心地将它从塑料盒中释放。切断孔和凝胶脚,并将凝胶放入转膜缓冲液中。将膜和凝胶夹在滤纸和海绵之间,制备转膜层。膜应靠近正极,凝胶应靠近负极。使用小滚筒去除凝胶和膜之间的气泡。夹住关闭的转膜箱,并将它浸入含有转膜缓冲液的转膜槽中。向外室加水,以保持系统冷却,并盖上盖子。打开电源,开始转移蛋白。时间和电压需要优化,请查看制造商的说明。现在蛋白已经从凝胶转移到硝酸纤维素膜上了,可以用抗体检测目标蛋白了。膜可以从盒中取出,现在应该可以看到分子量标志物了。如有需要,可以用丽春红 S 溶液对膜进行染色,从而确认蛋白质的转移。为了防止抗体发生非特异性结合,需要封闭膜。将封闭缓冲液倒在膜上,并置于摇床上轻轻摇动。通常情况下,需要使用 5% 牛奶或牛血清白蛋白(BSA)溶液在室温下孵育两小时或 4℃ 下孵育过夜。应优化封闭缓冲液的时间和类型,请查看您打算使用的一抗的数据表,了解详细信息。膜封闭后,去除封闭缓冲液,在同一溶液中加入稀释的一抗。与之前一样,置于摇床上孵育。通常会在室温下孵育一抗 1 小时或 4℃ 下孵育过夜。抗体浓度和孵育时间需要优化。如需任何指导,请查阅抗体数据表。倒出一抗,用洗涤缓冲液冲洗膜两次。随后在摇床洗涤膜 1 次,时长 15 分钟,再洗涤膜 3 次,每次 10 分钟。洗涤缓冲液通常是含 0.1% 吐温 20 的 Tris 缓冲盐溶液(TBS)或磷酸盐缓冲盐溶液(PBS)。倒掉洗涤缓冲液,在偶联二抗中孵育膜,二抗需先在封闭缓冲液中稀释。通常要在室温下孵育一小时,但抗体浓度和孵育时间需要优化。倒掉二抗,并按照上述步骤清洗膜。有几种不同的检测系统。如果二抗与酶偶联,则成像前,应在合适的底物中孵育膜。如果二抗是荧光偶联二抗,可以直接进入成像步骤。成像时使用 X 射线胶片或数字成像系统。将膜放入成像托盘中。将成像托盘放入成像系统。为了清楚地检测与目标蛋白相关的条带,可能需要优化曝光时间。
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实验员小哈&Western blot 关于Blocking(封闭)的一些心得 - 哔哩哔哩
哈&Western blot 关于Blocking(封闭)的一些心得 - 哔哩哔哩 实验员小哈&Western blot 关于Blocking(封闭)的一些心得实验员小哈关注专栏/实验员小哈&Western blot 关于Blocking(封闭)的一些心得实验员小哈&Western blot 关于Blocking(封闭)的一些心得
2020年08月24日 23:57--浏览 ·
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实验员小哈粉丝:10.2万文章:11
关注又来填坑了。在我的western blot操作视频中,使用了一个神奇的成品blocking buffer,室温15分钟,封闭就做完了,封闭效果也不错,关键是省时间。可是,如果实验室没有成品buffer可以用,怎么办?通常,是用1X TBST配制5%的脱脂牛奶,室温,1~2小时。或者用1X TBST配制5%的BSA,室温,1~2小时。封闭结束后,用5%的BSA配制一抗稀释液,4度,过夜。赶时间的时候,可以试试37度,封闭半小时。不赶时间,或者做到封闭这一步的时候,天色已晚想下班,可以4度封闭过夜。---------以下是trouble shooting讨论--------背景高,排除了抗体因素,高度怀疑是封闭不全的情况,解决策略:放弃省时间的想法,优先尝试4度封闭过夜这个条件。换用不同的封闭剂。背景过于干净,条带弱,排除其它因素,怀疑是封闭过度的情况,解决策略:将1X TBST换成不含Tween-20的1X TBS来配制封闭液。降低牛奶或BSA的浓度。减少封闭时间。(这条不是很推荐。)换用不同的封闭剂。不过,,,封闭过度的情况不是很常见,不应该作为条带弱的时候优先考虑的情况。今天还是要祝大家的Western blot顺利!欢迎提出、讨论更多的关于Western blot的问题~本文为我原创本文禁止转载或摘编
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Western Blotting Immunodetection Techniques | Bio-Rad
Western Blotting Immunodetection Techniques | Bio-Rad
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Life Science
Introduction to Western Blotting
Western Blotting Immunodetection Techniques
Western Blotting Immunodetection Techniques
Overview
Sample Prep
Electrophoresis
Transfer
Immunodetection
Image Acquisition
Image Analysis
Better Immunodetection
Information on antibodies, their selection, and use.
On This Page
Detection Overview
Blocking
Primary Antibodies
Secondary Antibodies
Detection Methods
Total Protein Detection
Tips & Protocols
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Detection Overview
After transfer, proteins from the gel will be immobilized on the membrane. This section describes the process of specifically detecting your particular protein of interest in order to identify and quantify its presence and abundance in the sample. Western blot detection of proteins utilizes primary antibodies that are specific for the target protein which are then in turn recognized by secondary antibodies that are conjugated with enzymes or fluorescent molecules for detection. The correct selection and use of appropriate primary and secondary antibodies is crucial for maximizing signal while reducing background. The proper use of protein loading controls are also critical for signal normalization in quantitative western blotting. We discuss the most popular methods to detect total protein on the membrane for loading controls and their relative advantages and disadvantages.
The Immunodetection Workflow
Western Blotting Antibody Detection
After proteins are transferred from the gel to the membrane, antibodies specific to your protein of interest (primary antibodies) are incubated with the membrane to allow them to recognize their targets. A second incubation with conjugated antibodies specific to the primary antibodies (secondary antibodies) enables visualization by various methods.
Block unbound membrane sites
Membranes are incubated in a blocking agent that masks any sites on the membrane that would allow antibodies to bind non-specifically. Failure to do so raises background.
Incubate with primary antibody. Wash away excess.
Membranes are then incubated with primary antibodies that are specific to your protein of interest. These bind to their targets and any excess is washed away.
Incubate with secondary antibody. Wash away excess.
The membrane is then incubated with secondary antibodies that recognize the primary antibodies and are also conjugated to either an enzyme or a fluoroscent molecule. Any excess is washed away.
Develop signal
For secondary antibodies conjugated with an enzyme, the membrane is incubated with a chemical substrate that produces a signal that can then be detected. If the secondary is conjugated to a fluorescent label, the membrane can be directly imaged in an instrument capable of fluorescent imaging.
Blocking
Following transfer, unoccupied antibody binding sites on the membranes must be blocked to prevent nonspecific binding. Failure to completely block these sites can lead to high backgrounds that obscure the signal.
Various blocking reagents are available, and no single blocker will be optimal for all antibody/antigen combinations. The best blocker for each experiment will depend on the antibody and membrane type. Optimize the detection system for maximal signal with lowest background by testing several blocking agents. Modern formulations like Bio-Rad’s EveryBlot Blocking Buffer are universal, performing well across a wide range of targets, sample types, and detection methods.
Blocking duration also affects the signal and background levels. Blocking for 1 hour with constant agitation is a good starting point. Excessive blocking times may result in lower sensitivity as epitopes can be masked by the blocking agent and proteins washed off the membrane. In contrast, insufficient incubation times will increase nonspecific binding of the primary antibody. Advanced formulations like Bio-Rad’s EveryBlot Blocking Buffer can complete the blocking step in 5 minutes.
For detection of a phosphorylated form of a protein, blocking buffers should not contain phosphorylated proteins to avoid high background signal. Avoid use of nonfat dry milk and instead use BSA, casein, or advanced formulations that are compatible with phosphoprotein detection.
Quick Tips:
Optimizing the Blocking Step in Western Blotting
Watch the video to understand the experimental parameters and which type of blocking agent to use to ensure success of your western blot.
Agent
Pros
Cons
Non-fat milk (1–5%)
Inexpensive, widely available
Not ideal for phosphoproteins, biotin-avidin/streptavidin, or AP detection
Speckling if not completely solubilized
BSA (0.5–5%)
Recommended for phosphoprotein detection, AP/biotin-avidin detection
Not usable with antibodies created using BSA-coupled peptides, phosphotyrosine Ab
Casein (1–2%)
Balance of stringency & sensitivity
Ideal for most detection methods
Blocking may be insufficient for complex biological samples
Gelatin (1–5%)
Inexpensive, widely available
Fish gelatin does not cross-react with mammalian proteins
Porcine gelatin solidifies in cold blocking
Speckling if not completely solubilized
Cannot be used with avidin-biotin detection
Specialized
Advantage depends on product
Can be expensive
Primary Antibodies
An antibody is an immunoglobulin protein such as lgG that is generated in response to exposure to a foreign substance, or antigen. Antibodies have specific affinity for the antigens that elicited their synthesis.
Primary Antibodies
Primary antibodies are raised against a protein of interest and will selectively recognize and bind to target proteins that have been immobilized to a membrane. Antibodies can be designed to be specific for certain parts of a target protein, or they can be designed to be specific for only modified versions of the protein.
Primary Antibody Selection
Antibodies should be specific, selective, and give reproducible results. To ensure the specificity of the primary antibody, it is important to use positive and negative controls when running your blot. As a positive control, purified proteins or lysates overexpressing the target protein can be used. As a negative control, you can include secondary-antibody-only controls (omitting the primary antibody incubation step) and samples from a tissue or cell lysates known not to express the target protein such as cells engineered with a genetic knockout. Before performing the experiments review the literature in order to understand the expression profile of the protein
Selecting Primary Antibodies for Best Results in Western Blotting
Choosing a Primary Antibody for Multiplex Western Blotting
How to Select the Right Antibody
Bio-Rad has developed tips to consider when choosing your antibody.
See Antibody Tips
The wording good and bad antibody or the most specific antibody should be avoided, since a specific antibody in one sample context can give rise to high cross-reactivity in another sample context depending on the nature of the epitope(s) that it will recognize.
— Edfors et. al. 2018, Nature 9:4130
When selecting a phospho-specific antibody for your experiments, it is crucial to ensure that the antibody specifically detects the protein of interest only when it is phosphorylated at the indicated site. You might also want to consider treating cells with growth factors or chemical compounds that induce or inhibit expression of the target.
Monoclonal vs. Polyclonal Antibodies
The antibodies used to detect the target protein in a western blot will be either monoclonal or polyclonal. Polyclonal antibodies consist of a mixed pool of immunoglobulin molecules that bind to several different epitopes found on a single antigen. Polyclonals are usually produced in rabbits, donkeys, sheep, and goats, and are purified from serum.
In contrast, monoclonal antibodies bind to a single epitope within a target antigen. They are composed of homogeneous cloned immunoglobulin molecules, rather than the heterogeneous antibody mixture typical of polyclonals. Monoclonals are made by fusing antibody-producing cells from the spleen of the immunized animal (usually a rat or mouse) with an immortalized cell line to produce single specificity antibodies that can be purified from tissue culture supernatant.
Monoclonal
Polyclonal
Specificity
Specificity for a single epitope.
Varying specificities to multiple epitopes
Identification
Identifies whether a particular region of a protein is present
Identifies the entire target protein via binding at multiple sites. Since multiple epitopes are targeted, there is a higher likelihood of detection of the target
Cross-Reactivity
May cross-react with other proteins that share this epitope, such as isomers or common motifs
Higher background and cross-reactivity possible due to detection of multiple epitopes, any of which may be shared by related proteins
Sensitivity
Usually less sensitive since only a single antibody molecule binds to each target
More sensitive because signal is amplified through the binding of several antibodies per target
Cost
More expensive to produce initially, but available in an unlimited supply
Less expensive to produce initially, but supply is limited to immunized animal(s). Greater variability between preparations
Primary Antibody Incubation
After blocking, the membrane is incubated in a solution containing the primary antibody, usually diluted in blocking buffer. The time and temperature of incubation depends on the binding affinity of the antibody to the target protein and should be determined for each antibody individually. One hour at room temperature with gentle agitation is a good starting point. In order to reduce the background staining, the amount of Tween 20 used in the buffers is also important.
Antibody Concentration
The optimum antibody concentration is the dilution of antibody that still yields a strong positive signal without background or nonspecific reactions. Instructions for antibodies obtained from a manufacturer typically suggest a starting dilution range. For custom antibodies or for those where a dilution range is not suggested, good starting points are:
1:100–1:1,000 dilution when serum or tissue culture supernatants are the source of the primary antibody
1:500–1:10,000 dilution of chromatographically purified monospecific antibodies
1:1,000–1:100,000 dilution may be used when ascites fluid is the source of antibody
Quick Tips:
How to Optimize Primary Antibody Concentration and Incubation for Western Blots
Watch this video to explore considerations for optimizing incubation time and dilution of primary antibodies when performing a western blot.
See Our Western Blotting Primary Antibodies
Phospho-Specific Antibodies
Protein phosphorylation, the addition of a phosphate group to serine, threonine or tyrosine residues, is an important cellular process utilized to send cellular signals from the membrane to the nucleus. Protein phosphorylation may result in conformational changes that trigger the activation or inactivation of an enzyme.
Phospho-specific antibodies are primary antibodies that detect only the phosphorylated forms of proteins and recognize the phosphorylated serine, threonine, or tyrosine residues in the context of the rest of the protein. Using a combination of total protein detection (using primary antibodies that recognize both phosphorylated and non-phosphorylated forms of the protein) and phospho-specific antibodies, it is possible to assess the degree of phosphorylation for any given protein.
Prior to using phospho-specific antibodies, ensure that the antibodies have been validated for your experimental system. This can be accomplished by using positive and negative controls. For example, lysates from cells that are either untreated or have been treated to stimulate the pathway of interest can act as negative and positive controls. Likewise, absence of detection can act as a negative control on lysates that have been treated with lambda phosphatase to remove phosphate groups.
See Our Phospho-Specific Antibodies
Secondary Antibodies
Purification of Cross-Adsorbed Antibodies
A solution of secondary antibodies raised against mouse lgG is passed over a column containing immobilized serum proteins from potentially cross-reactive species such as rat or rabbit.
Only antibodies highly specific for mouse lgG will flow through the column, while antibodies cross-reacting to rat or rabbit will bind and remain in the column. The flow-through solution contains antibodies that specifically recognize mouse lgG.
Secondary antibodies are specific for the isotype and species of the primary antibody. For example, a goat anti-rabbit secondary is an antibody raised in goats against a primary antibody raised in rabbits.
Secondary antibodies bind to a number of different conserved regions on the primary antibody, and act to amplify the signal, increasing detection sensitivity. Secondary antibodies are labelled with either an enzyme for colorimetric or chemiluminescent detection or with a fluorescent dye for fluorescent detection of the protein of interest.
Cross-Adsorbed Antibodies
Secondary antibodies raised against primary antibodies of one species can recognize those from other species, especially if they are from closely related animals. For example, parts of the constant regions of mouse and rat antibodies are evolutionarily conserved, leading to conserved epitopes between both species. Since secondary antibodies are generally polyclonal, when generating secondaries against mouse, a subset of those secondaries will be specific for those conserved epitopes, being able to recognize them whether the epitope resides on a mouse or rat antibody. This cross-reactivity could lead to unexpected bands when multiplexing or cause high background.
To remove the undesired antibodies from a polyclonal pool, an affinity chromatography column is used to purify the secondary antibody preparation of offending cross-reactive species. Clonal antibodies that recognize the immobilized antigen(s) are removed. The unbound pool of antibodies is now cross-adsorbed against the immobilized antigen and should show no reactivity towards it.
The most common type of cross-adsorbed secondary antibody is species specific, which is most useful for multiplexing, although there are also instances when isotype-specific antibodies are required. For example, immunization with a purified IgG1 preparation might be expected to generate a serum with a specific reactivity towards IgG1 and no cross-reactivity with other antibody isotypes. However, due to the polyclonal nature of the serum combined with the conservation of some epitopes between classes and isotypes, a component of the polyclonal serum may react with an epitope on IgG1 that is also found on IgG2a, confounding the isotype specificity.
A solution of secondary antibodies raised against mouse IgG is passed over a column containing immobilized serum proteins from potentially cross-reactive species such as rat or rabbit.
Only antibodies highly specific for mouse IgG will flow through the column, while antibodies cross-reacting to rat or rabbit will bind and remain in the column. The flow-through solution contains antibodies that specifically recognize mouse IgG.
Secondary Antibody Concentration
Similar to primary antibodies, the optimum antibody concentration is the dilution of antibody that still yields a strong positive signal without background or nonspecific reactions. Fortunately, high-quality secondary antibodies are commonly available, and manufacturers typically suggest a starting dilution range.
See Our Western Blotting Secondary Antibodies
Quick Tips: How to Choose Secondary Antibodies for Multiplex Western Blotting
In this video, we discuss the best practices for selecting secondary antibodies that are compatible with your primary antibodies and your detection methods to produce high-quality, reproducible results.
Detection Methods
In the past, many different methods were used for western blot detection, but now the vast majority employ enzymatic chemiluminescence or fluorescent detection. Thus, most secondary antibodies are conjugated to an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) for use with a chemiluminescent substrate or labeled with a fluorescent compound for imaging.
Chemiluminescence Detection
Chemiluminescence is a chemical reaction in which the oxidation of a chemical substrate such as luminol is catalyzed by an enzyme, typically horseradish peroxidase (HRP), and the reaction produces light as a byproduct. The resulting light can be captured on film or by a digital imager.
Luminol emits light only weakly, so enhancers are added to the reaction to increase the signal. This enhancement of a luminol-based signal is commonly referred to as enhanced chemiluminescence (ECL). There are a number of different enhancers available, some of which can increase the signal by as much as a 1,000-fold, making ECL more sensitive than other common detection systems such as conversion of substrates to colored precipitates.
The light intensity will be roughly proportional to the quantity of your protein of interest, allowing semi-quantitation of relative protein abundance. Since this detection method relies on an enzyme-substrate interaction, the kinetics of the reaction plays a role in the linearity of the signal. Therefore, for more accurate quantitation, care must be taken to ensure that the sample load is within the linear range of the assay and the detection signal is not saturated.
Luminol oxidized by HRP in the presence of H2O2 leads to the formation of a 3-aminophthalate dianion and the release of light.
Quick Tips:
How to Image a Chemiluminescent Western Blot
Watch this video to see how to prepare a western blot membrane for chemiluminescent detection and ensure a strong chemifluorescent signal during image acquisition.
See Our Chemiluminescent Substrates »
Fluorescence Detection
In fluorescence detection, a primary or secondary antibody is labeled with a fluorescent molecule (a dye or fluorophore). A light source that produces photons within the fluorophore's excitation spectrum excites the fluorophore, and the fluorophore then emits photons with a longer wavelength. The difference in wavelength between excitation and emitted light is termed Stokes shift. This emitted light of longer wavelength can then be distinguished from the excitation light via appropriately designed optical filters and detected by a digital imager.
Fluorescence detection has a number of advantages over traditional chemiluminescent detection. As fluorescent detection does not rely on an enzyme-substrate reaction, the use of fluorophores can be more quantitative than chemiluminescent detection, is often faster, and reduces waste.
The greatest advantage of fluorescent western blotting detection over chemiluminescent detection is the ability to simultaneously detect a large number of proteins on one blot, using a process called multiplexing. This relies on choosing fluorophores that fluoresce at different wavelengths and can be distinguished by the digital imager.
Fluorophore Selection
When using fluorescence detection, consider the following optical characteristics of the fluorophores to optimize the signal:
Quantum yield — efficiency of photon emission after absorption of a photon. Processes that return the fluorophore to the ground state but do not result in the emission of a fluorescence photon lower the quantum yield. Fluorophores with higher quantum yields are generally brighter.
Extinction coefficient — measurement of how well a fluorophore absorbs light at a specific wavelength. Since absorbance depends on path length and concentration (Beer's Law), the extinction coefficient is usually expressed in cm -1 M-1. As with quantum yield, fluorophores with higher extinction coefficients are usually brighter.
Stokes shift — the difference in the maximum excitation and emission wavelengths of a fluorophore. Since some energy is dissipated while the fluorophore is in the excited state, emitted photons are of lower energy (longer wavelength) than the light used for excitation. Larger Stokes shifts minimize overlap between the excitation and emission wavelengths, increasing the detected signal.
Excitation and emission spectra — excitation spectra are plots of the fluorescence intensity of a fluorophore over the range of excitation wavelengths; emission spectra show the emission wavelengths of the fluorescing molecule. Choose fluorophores that can be excited by the light source in the imager and that have emission spectra that can be captured by the instrument. When performing multiplex western blots, choose fluorophores with widely separated emission spectra to enable good signal separation among the different color channels.
Fluorophore Stokes Shift
A high-energy photon excites a fluorophore, causing it to leave the ground state (S0) and enter a higher energy state (S11). Some of this energy dissipates, allowing the fluorophore to enter a relaxed excited state (S1). When the fluorophore returns to the ground state, a photon of light is emitted. Since some of the energy was dissipated, the emitted photon is of lower energy (longer wavelength).
The difference in wavelength between the exciting light and the emitted light is called the Stokes shift. Larger Stokes shifts minimize the overlap between the excitation and emission wavelengths, increasing the detection signal-to-noise ratio.
See Our Products for Fluorescent Western Blotting Detection »
Total Protein Detection
Total protein staining provides an image of the complete protein pattern on the blot. This information helps determine transfer efficiency and molecular weight of the transferred proteins. This information can also be used to determine relative quantity of sample that was loaded in each lane.
Several total protein stains are available and commonly used in western blotting. The table below provides an overview of total protein detection methods and applications.
Total Protein Detection Methods for Western Blotting
Detection Method
Sensitivity
Advantages
Disadvantages
Imaging
Anionic dyes (Ponceau S, Fast Green, Coomassie Brilliant Blue, Amido black)
100–1,000 ng
Inexpensive, rapid
Coomassie and Amido black are not compatible with downstream immunodetection
Epi illumination
Fluorescent stains (SYPRO Ruby and Deep Purple)
2–8 ng
Sensitive; most are compatible with immunodetection
Additional staining and destaining steps
Fluorescent capable digital imager with UV, visible light LED, or lasers
Stain-Free Imaging
2–28 ng
Rapid and convenient—ready in less than one minute, and no separate staining or destaining required. Sensitivity comparable to Coomassie
Requires use of Stain-Free Gels and compatible imager
Bio-Rad imagers
Stain-Free Imaging Technology
Bio-Rad's stain-free technology allows direct visualization, analysis, and documentation of protein samples in PAGE gels and on blots, without staining or destaining. Stain-free imaging provides equal or better sensitivity compared to Coomassie staining and eliminates organic waste disposal concerns.
Linear dynamic range provided by stain-free technology for total protein measurements. HeLa cell lysate dilutions from 80–2.5 µg total protein.
Learn More about Stain-Free Imaging Technology »
Total Protein Normalization
Bio-Rad’s stain-free technology can be used for total protein normalization of western blots. Total protein normalization uses the signal of all proteins in a sample to determine load. This approach is more robust against expression changes of any one protein to an experimental condition, which can occur when using a single “housekeeping” protein.
Learn More about Total Protein Normalization »
Immunodetection Tips and Protocols
Western Detection Tips
Use high-quality primary antibodies.
A good antibody is sensitive, meaning it can detect low amounts of your target, and is also specific, recognizing only the target, without giving spurious secondary bands. Antibody vendors are increasingly offering antibodies that have been certified for western blotting.
Use cross-adsorbed secondary antibodies.
Cross-adsorbed antibodies offer a lower chance of cross-reactivity and reduced background giving more reliable results. They are readily commercially available so they should be used whenever possible.
Optimize antibody concentration to maximize signal to background.
Sensitive detection of your target depends on high signal and low background. Reducing antibody concentration can reduce desired signal, but the greater reduction of background more than makes up for this.
For multiplex detection, first optimize antibodies to targets individually.
When detecting multiple targets simultaneously, carry out detection of each target individually first. This will simplify any needed troubleshooting.
Western Detection 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.
Stain-Free Western Blotting Guide
Find out how Stain-Free technology can revolutionize your western blotting.
Better Western Blotting Guide
Tips, Techniques, and Technologies from the Western Blotting Experts at Bio-Rad Laboratories.
Protein Blotting Guide
(PDF 7.2 MB)
Details on blotting technology, methods, products, tips, techniques, and troubleshooting guidelines.
Western Blot Doctor
Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.
Best Practice for Western Blot Detection of Phosphorylation Events
Ten tips to ensure robust data generation and cleaner blots.
Protocol: Detection of Phosphorylated Proteins by Western Blotting
This protocol describes how to detect phosphorylated proteins by western blotting using Phospho-Specific PrecisionAb Antibodies.
Fundamentals of Western Blotting Course #3: Immunodetection
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.
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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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