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
血泪教训,Western blot史上最全避雷手册。 - 知乎首发于Western blot踩雷和制作大全切换模式写文章登录/注册血泪教训,Western blot史上最全避雷手册。聊点学术AI病理+量化病理方案设计提起Western blot (WB),很多人都能哭诉半宿。这是一个基础实验,也是一个让大家头疼的实验,简称“玄学”。话不多说,做过的都懂。今天给大家提供WB最全避雷手册,希望大家能愉快实验,顺顺利利。倾 尽 全 力,长 文 预 警1、WB有什么优点?答: 灵敏,可达ng级,用ECL显色法可达pg级。灵敏,相对便宜且特异性高。2、为什么我的细胞提取液中没有目标蛋白?答:a) 你的细胞中并不表达这种蛋白质,换一种细胞或查文献弄清楚; b)你的细胞中的蛋白质被降解掉了,你必需加入PMSF,抑制蛋白酶活性; c) 你的抗体不能识别目的蛋白,检查抗体说明书,看是否有问题。3、我的细胞提取液有的有沉淀,有的很清亮,为什么呢?答:a)有沉淀可能因为你的蛋白没有变性完全,可以适当提高SDS的浓度,同时将样品煮沸时间延长,一般需96℃以上10-15min左右; b) 也不排除你的抗原浓度过高,这时需再加入适量上样缓冲液。4、我做的蛋白质分子量很小(10KD左右),请问怎么做WB?答:尽量选择0.2μm的膜,缩短转移时间;也可将两张膜叠在一起再电转。5、我的目的带很弱,怎么加强?答:最主要是加大抗原上样量;同时也可以将一抗稀释比例上调。6、胶片背景很脏,有什么解决方法?答:减少抗原上样量,降低一抗浓度,改变一抗孵育时间和温度(尽量4℃过夜,此孵育条件比37℃1h要温和很多),并提高封闭液浓度。7、目标带是空白,周围有背景,是为什么?答:你的一抗浓度较高,二抗上HRP催化活力太强,同时你的显色底物处于一个临界点,反应时间不长,将周围底物催化完,形成了空白即“反亮现象”。将一抗和二抗浓度降低,或更换新进口的显色底物。8、我的胶片是一片空白,是怎么回事?答:如果能够排除下面的几个问题那么问题多半出现在一抗和抗原制备上。a) 二抗的HRP 活性太强,将底物消耗光;b) ECM底物中本身含双氧水,不稳定,见光或温度高时易失活;现配现用。c) ECL底物没覆盖到相应位置;d) 二抗时间过期,或平时使用不当导致二抗失活。9、我在显影液中显影1分钟和5分钟后,底片漆黑一片,是什么原因呢?答:a) 可能是红灯造成的, 胶片本来就被曝光了,可以在完全黑暗的情况下操作,看是否有改善.;b) 显影时间过长,一般3-5分钟就够了,特殊情况需摸索显色时间。10、DAB好还是ECM好?答:DAB有毒,但是比较灵敏,是HRP最敏感的底物;ECM结果容易控制,但被催化时灵敏度差一点,但如果达到检测的阀值,就特别灵敏,可以检测pg级抗原。具体选择还是要看你实验的情况,目前大家一般使用ECM。11、抗原检测出的分子量比资料上的大,是怎么回事?答:a)所检测的抗原形成二聚体。增多巯基乙醇量,煮沸变性时间延长,可以打开二聚体。b)蛋白本身有修饰如糖基化、磷酸化等均会增大蛋白分子量。12、蛋白转移不到膜上,但胶上有,同时Marker 转上去了,为什么?答:可能是:a)样品浓度过低;b)转移时间不够。(注意!Marker并不是蛋白是否转移到膜上的标准,它只是为我们提供简单的指示作用。)13、磷酸化抗体的检测样本制备时是否一定要加NaF等?答:NaF是一种广谱磷酸化酶的抑制剂,一般最好加。但是不加也可以,大部分时候是不用加的。建议可多种抑制剂混合使用。14、要验证某个细胞上有无该蛋白的存在,需要做免疫组化和WB试验吗?做这两个试验时的一抗和二抗可以共用吗?答:a)免疫组化可以用来进行定位,但是不能精确定量,而且有时会有假阳性,不易与背景区分;WB可以特异性检测某个蛋白质分子,如抗体选择合适,灵敏度很高。WB进行定量,但是不能定位。b)两种实验的一抗有时候不能通用,公司的产品说明一般都会说明可以进行什么实验,在免疫组化时,是否适合石蜡切片或者冰冻切片。15、做WB时,提取蛋白后冻存(未加蛋白酶抑制剂),用了一抗,开始还有点痕迹,现在越来越差,上样量已加到120μg,换了一种一抗仍不行。是什么原因?蛋白酶抑制剂单加PMSF行吗?答:怀疑是样品问题,可能是:a)样品不能反复冻融,建议制备好样品后混匀,小体积分装;b)样品未加蛋白酶抑制剂。同时,建议检查WB过程,提高一抗浓度。对于加蛋白酶抑制剂来说,一般加PMSF就可以了,最好能多加几中种蛋白酶抑制剂(PMSF在水溶液中不稳定,30min就会降解一半,必须在每一分离和纯化步骤中加入新鲜的PMSF,样品处理超过1小时,补加1次。见水易分解,使用前加入)。16、细胞水平要做WB,多少细胞提的蛋白够做WB?答:一般5×10^6就足够了;特殊情况需根据自己的研究来确定。17、同一样品能同时提RNA又提蛋白么?这样对WB有无影响?答:能,完全没有问题;这是两个不同的实验。18、同一蛋白样品能同时进行两种因子的WB检测吗?答:当然可以,有的甚至可以同时测几十种样品;有时如果抗体特异性高且效价高,同时使用2种一抗,可在一张膜上检测2种不同蛋白。19、如果目标蛋白是膜蛋白或是胞浆蛋白,操作需要注意什么?答:如果是膜蛋白和胞浆蛋白,所用的去垢剂要温和得多,这时最好加上NaF去抑制磷酸化酶的活性;部分膜蛋白不好提取,尤其是亚细胞器的膜蛋白。可考虑最新的invent柱式提取法,提取效率高,蛋白丢失较少。20、我的样品的蛋白含量很低,每微升不到1微克,但是在转膜时经常会发现只有一部分蛋白转到了膜上,就是在转膜后染胶发现有的孔所有的蛋白条带都在,只是颜色变淡了,有什么办法可以解决?答:你可以加大上样量,没有问题,还有转移时你可以减少电流延长时间,加20%甲醇;还有一种办法就是采用最新的WES来检测,灵敏度极高,但价格贵。21、想分离的蛋白是分子量200kd的,SDS-PAGE电泳的分离胶浓度多大合适?积层胶的浓度又该用多少?这么大分子量的蛋白容易作WB吗?答:200kd的蛋白不好做,分离胶用7%,积层胶 3.5%。这种分子量蛋白建议加大电泳电转的电场强度和加长作用时间,但必须控制温度,保证低温电泳和电转。22、如果上样量超载,要用什么方法来增加上样量?答:可以浓缩样品,也可以根据你的目标分子量透析掉一部分小分子蛋白。一般地,超载30%是不会有问题的,建议尽量不超载加样。如果已经超了不少了,而且小分子量的也要,可以考虑加大胶的厚度,以增大上样孔体积,可以试1.5mm厚的胶。23、蛋白变性后可以存放多久?答:-80℃,若没有反复取出冻融,保存一两年没有问题。最关键两条:不要被蛋白酶水解掉;不要被细菌消化掉(也是被酶水解了)。24、我所测定的蛋白分子量是100KD左右,按理说分离胶应当采用7.5%,但资料却要求分离胶和浓缩胶均采用11%的配方,不知为何?答:上述提到的两种凝胶均可以使用,因为105KD的蛋白在上述两种胶的线性分辨范围内,但需注意目的条带位置肯定会发生一定地偏移。25、二抗是生物素化的抗体,三抗是亲和素生物素体系,不知采用这样的方案后,封闭液是否要作调整,能否再用5%的脱脂奶粉呢?好像有资料说脱脂奶粉会影响亲和素生物素的生成,是吗?答:不能使用脱脂奶粉,因为脱脂奶粉中含较多的生物素,用BSA(5%、8%)代替会好一点。26、一般一次上样的蛋白总量是多少,跟目的蛋白的表达量有关系吗?答:WB一般上样30-100μg不等,结果跟目的蛋白的表达丰度、上样量、一二抗的量以及孵育时间都有关系,也与显色时间长短有关。开始摸条件时,为了拿到阳性结果,各个步骤都可以量多一点时间长一点,当然背景也就出来了。要拿到好的结果,如果抗体好的话比较容易,抗体不好的话就需要反复地试了。当然有的蛋白不适合WB的怎样做也不行,或者抗体效价低则注定事倍功半。27、做组织样品的WB的时候,怎样处理样品?答:必须进行研磨、匀浆、超声处理(注意降温),蛋白质溶解度会更好,离心要充分(12000转/min,10-15min)。膜蛋白必须用更剧烈的方法抽提,低丰度膜蛋白可能还要分步抽提(超速离心)。还有一点就是组织中的蛋白酶活性更强,需要注意抑制蛋白酶的活性(加入足量的PSMF)。28、大分子量蛋白200KD或以上,在做WB要注意什么呢?答:做200KD蛋白的WB时要注意,分离胶最好选择>7%的;胶很软,剥胶时要小心;转移时间需要相应延长(至少300mA,3h或以上);必须要使用大量程的Marker(否则出现杂带不知如何分析问题)。29、有什么方法可以提高上样量?答:a)可以浓缩样品;或增大上样体积来增大上样量;b)用5X的上样缓冲液来稀释变性。30、蛋白的上样量有没有什么具体的要求?答:上样量要根据实验的要求来定,如果要求是定量和半定量的WB则上样量要均等,如果只是要定性,则没有太大的关系, 尽量多上就行了,但是不要超载。31、一抗,二抗的比例是否重要?答:比较重要,调整好一抗,二抗的比例,可以去掉大部分的非特异性底色。32、要做磷酸化某因子WB,其二抗有何要求?答:主要是一抗要选择好。对二抗无要求,要看你实验条件来选择,一般推荐用HRP标记的二抗。33、免疫组化和WB可以用同一种抗体吗?答:免疫组化时抗体识别的是未经变性处理的抗原决定簇(又称抗原表位),有些表位是线性的,而有的属于构象型;线性表位不受蛋白变性的影响,天然蛋白和煮后的蛋白都含有;构象型表位由于受蛋白空间结构限制,蛋白煮后变性其构象会消失,仅剩下一级结构。如果你所用的抗体识别的是蛋白上连续的几个氨基酸,也就是线性表位,那么这种抗体可同时用于免疫组化和Western,而如果抗体识别构象形表位,则只能用于免疫组化。一般我们做的时候没有这么复杂的考虑,多看看抗体说明书所注明的实验范围来做,这样省时省力。34、WB中抗体的可以重复应用吗?答:所有的抗体工作溶液一般不主张回收冻存反复使用,但是如抗体比较珍贵,可反复使用2-3次。稀释后应在2-3天内使用,4度保存,避免反复冻融。35、在做WB时,PVDF膜用甲醇浸泡的目的?答:PVDF膜用甲醇泡的目的是为了活化PVDF膜上面的正电基团,使它更容易跟带负电的蛋白质结合,做小分子的蛋白转移时多加甲醇也是这个目的。36、检测同一抗体的磷酸化和非磷酸化的抗原可以在同一张膜上吗?答:肯定可以,建议先检查非磷酸化蛋白,然后使用抗体洗脱液将抗体洗去,再在该膜上重新孵育磷酸化一抗。37、转膜后经丽春红染色的条带,为什么大蛋白分子的一端的转膜好象不是很好,为什么?答:这是正常的,大分子的蛋白转移很慢,你延长转移时间和电流,大分子一端就会好的多,但是小分子的就有可能会变淡(转过的表现)。注意一点:丽春红染色较好,不是你的目标蛋白成功转移至膜上的标准,这是两个概念,;丽春红染色很多时候只是提供一个粗略的参考而已。38、做WB时,同样的抗体在免疫组化能做出,而WB却不能?答:这多半是抗体的问题,要看抗体的说明,是否能做WesternBlot和免疫组化。39、如果是6×8转印膜,要加多少一抗?答:一抗的稀释度是有说明的,根据你的一抗看看就知道了,但是那么大的膜孵育体积一般最少为3-5ml。建议尽量还是裁剪膜,不要整张膜孵育,费抗体而且可能会孵育不均匀(个人建议,仅供参考)。40、上下槽缓冲液有何要求,怎样才能达到最佳效果。答:无特殊要求。但我们一般是上槽放新鲜的缓冲液,下槽可以是重复使用过一两次的缓冲液;小编发现电泳时产热也很厉害,其实可以在电泳时加冰浴。41、跑电泳的时候配的胶总是“缩”是什么原因呢?是有的成分不对吗?答:没有问题,就是你胶里的水分被电泳产热蒸发了。其次配好的胶在过夜时拿保鲜膜包起来,在里面加点电泳液保持湿度就可以了;也可能30%聚丙酰胺有问题,你可以重新配制一份试试或直接买商用的30%聚丙酰胺;能够替换的试剂,尽量换一下,选用进口试剂。42、蛋白质的分子量跨度很大,如要分离小21KD,中至66KD,大至170KD,可以一次做好吗?答:这么广的分布不好转移,一般建议:21KD和66KD可以一起转,一起做。采用12%SDS-PAGE,湿转120mA,45-60min就可以了,可以根据你实验室师兄师姐的调节;而170KD蛋白用7%SDS-PAGE,至少200mA 120min。43、不能很好地将大分子量蛋白转移到膜上,转移效率低怎么样解决?答:可以考虑:转移缓冲液中加入20%甲醇(是指终浓度),因为甲醇可降低蛋白质洗脱效率,但可增加蛋白质和NC膜的结合能力,甲醇可以防止凝胶变形,甲醇对高分子量蛋白质可延长转移时间;转移缓冲液加入终浓度0.1%SDS,也是为了增加转移效率;用优质的转移膜,或使用小孔径的NC膜(0.2μm)。44、我用的是可视Marker,但是电泳总跑不全8条带,请问什么原因?怎样改善?胶用过8%,10%,12%,都是这样。Marker是新买的。答:一般来说,是小分子量Marker跑走了,增加胶浓度或减少电泳时间试试看。当然梯度胶也是不错的选择,现在已经有商用梯度胶了,大家可自行选择。45、是否WB实验半定量一定要加内参?答:对于发表文章的实验一定要加内参,实验严谨,证据充分。46、核内抗原WB内参选择什么合适?答:可以选用组蛋白,组蛋白在细胞核中的表达是很稳定的,有很多都可以当成内参,动动手查查就可以选出你要的内参。(也可以私信小编哦)47、做半定量WB,内参β-actin,GAPDH哪个好?答:一般选用β-actin就可以,也可以使用GAPDH,但是如果做能量、代谢这方面的研究还是尽量选择β-actin。48、想问一下细胞裂解液选择蛋白酶抑制剂时有什么原则吗?受不受组织来源的影响?胞膜和胞浆有区别吗?答:一般来说提取时加入广谱的蛋白酶抑制剂就可以了,操作时保持冰浴,保持低温。除非有文献特别指明必须使用特殊方法,一般来说没有区别。49、转膜后的脱脂奶粉封闭液是5%的TBST脱脂奶粉”,其中TBST的T是Tween吗,浓度是多少?答:T就是Tween,全称Tris-Buffered Saline Tween-20 (TBST),组成:8g NaCl,2.42g Tris base,加水800mL充分溶解, 加 500-1000μL 的 Tween-20,用HCl 调节pH 至 7.4,加水定容至 1L。50、封闭,一抗,二抗时的温度有没有什么规定呢,比如在室温里做,或者要在4度下?答:均可在室温进行,如果时间不够,一抗孵育可以先在室温进行一个小时,然后4度过夜。小编建议尽量还是4℃过夜孵育,原因在上面已说明。51、请问一下PVDF膜和硝酸膜结合蛋白的原理是什么?答:一般而言,硝酸纤维素膜是通过疏水作用来和蛋白质相联,这样的话,反复洗几次后,蛋白容易掉下来,结果较差。PVDF膜主要通过它膜上的正电荷和蛋白接合,同时也有疏水作用,但相对较弱。这样的话,PVDF膜和蛋白接合较牢,不易脱落,结果较好。52、怎样才能跑出漂亮的胶,泳道都能很直,上样的量和灌胶是否很重要,电压有什么要求?答:影响跑胶跑的质量,提供2个小建议:a)小电压会使胶的分子筛效应得到充分发挥。电压越小,条带越漂亮,浓缩胶55V,分离胶75V就能跑得很好,但是实验的时间会很长。b)胶的均匀度,胶越均匀,条带越窄,分离越均匀。倒胶之前,一定要充分混匀(不要有气泡),玻璃板一定要干净,双蒸水隔离时,一定要比较轻地加上去,避免稀释上层的分离胶。53、为什么提高大分子量蛋白的转移的时候,小分子量蛋白会丢失一些哪?什么原因?答:小分子的蛋白在转移过程中,会透过膜去,所以大分子的上去以后,有一部分小分子的就透过去了。54、电泳中常出现的一些现象:①“︶” 条带呈笑脸状原因:凝胶不均匀冷却,中间冷却不好;电泳系统温度偏高,而且电场强度太大(电泳的电场大致呈现U形,所以因为赶时间而加大电压可能会出现这个)。②“︵” 条带呈皱眉状原因:可能是由于装置不合适,特别可能是凝胶和玻璃挡板底部有气泡,或者两边聚合不完全。③条带拖尾原因:样品溶解不好,或存在一定程度地蛋白降解。④纹理(纵向条纹)原因:样品中含有不溶性颗粒,可能抽提蛋白时出了问题。⑤条带偏斜原因:电极不平衡或者加样位置偏斜,一定要子平面台子上电泳,胶要做好。⑥条带两边扩散原因:加样量过多,弥散至孔周围了,减少上样量或者使用5X上样缓冲液。55、WB结果中背景较高可能的原因及建议:①膜封闭不够,建议延长封闭的时间;选择合适的封闭液。②一抗稀释度不适宜,太高,选择最适宜的抗体稀释度。③ 一抗孵育的温度偏高,建议4℃过夜孵育。④选择的膜容易产生高背景,一般NC膜的背景会比PVDF膜低,但是NC膜吸附力也就差了一点。⑤膜在整个实验过程中干过或手套反复接触过,实验过程中要注意保持膜的湿润,使用镊子夹取膜。⑥检测时曝光时间过长,可减少曝光时间。56、WB结果中杂带较多可能的原因及建议:①目的蛋白存在多个修饰位点(磷酸化位点、糖基化位点等等),本身可以出现多条带。查文献或生物信息学分析,获得蛋白序列的修饰位点信息,去除修蛋白饰后确定蛋白实际大小,这个需要一定的生物化学基础和生信分析能力。②目的蛋白有其它剪切本,查阅文献或生物信息学分析其可能性。③样本处理过程中目的蛋白发生降解,加入蛋白酶抑制剂;样本处理在冰上操作。④上样量过高,过于敏感,适当减少上样量。⑤一抗特异性不高,重新选择或制备高特异性的抗体。⑥一抗不纯,纯化抗体。⑦一抗或者二抗浓度偏高,适当降低抗体浓度。57、WB结果中无信号或显示信号弱可能的原因及建议:①你所检测的样本中不表达目的蛋白,选择表达量高的细胞作为阳性对照,用于确定检测样本是否为阴性,同时查阅文献。②检测样本低表达目的蛋白,提高上样量,裂解液中注意加入蛋白酶抑制剂。③转移不完全或过转移,可以用丽春红染膜并结合染胶(考马斯亮蓝)后确定条带是否转至膜上或转移过头;适当调整转膜的时间和电流。④抗体不能识别测试种属的相关蛋白,购买抗体前应当认真阅读抗体说明书,确定其是否能够交叉识别测试种属的对应蛋白。⑤一抗孵育时间不足,建议4℃结合过夜。⑥二抗与一抗不匹配,选择针对一抗来源的种属的抗体。⑦洗膜过度,洗膜时间不宜过长,加入的去垢剂不宜过强或过多,建议使用0.1%的弱去垢剂Tween-20。58、其它现象:①膜上多处出现黑点或黑斑,原因有2点,抗体与封闭试剂发生非特异性的结合;或者配的奶粉没有充分溶解,奶粉团粘在膜上而没洗干净。②反白(条带显白色),原因一般是目的蛋白含量太高或者一抗浓度偏高。③蛋白分子量偏低或偏高,原因:胶浓度不适合,高分子量要用低浓度胶;小分子蛋白要用高浓度胶。59、安全爱护身体,保护自己。安全第一,实验第二。安全无小事!操作有毒试剂时,一要定带手套和口罩,且操作挥发性试剂一定要在通风橱中进行。This is the dividing line.以上为本期内容。简单地为大家提供一些建议,希望大家的实验能顺顺利利,早日完成SCI大业。发布于 2020-09-03 08:58蛋白质分子生物学实验赞同 119757 条评论分享喜欢收藏申请转载文章被以下专栏收录Western blot踩雷和制
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 ·
--喜欢 ·
--评论
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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Wb经验帖‖page5 封闭的细节
(如果最近经验有反响,我可以写ip/coip、细胞经验帖)
2-3mm高度的牛奶即可,不要多了,不要多了,不要多了,不要多了!!!!因为牛奶多了,加上你牛奶封闭时间不够的话,就会封闭效果差,导致最后背景黑差。(想象一下,你一瓶水晃动,和半瓶水晃动,哪个同等
丁香园-
基础科研-蛋白质和糖学-
2022-07-21 10:58:41
WB电泳脱脂奶封闭时间
一般转膜完毕以后,用脱脂奶封闭多久比较合理啊,是不是必须达到2个小时呀。还是说和蛋白的分子量有关呀。
丁香园-
基础科研-蛋白质和糖学-
2021-01-07 11:48:08
请教一个WB的时间问题
由于专硕白天要上临床,实验时间限制,不能一天做完WB,我能第一天晚上做到封闭抗体,4°过夜至第二天晚上,然后第二天晚上接着孵一抗、二抗、显影吗?(我看有些资料上是说封闭抗体室温摇床1h或者4°过夜
丁香园-
基础科研-蛋白质和糖学-
2022-07-26 09:14:58
WB曝光时间控制在多长合适
WB 曝光时间控制在多长时间内合适呢,比如有的蛋白2、3S就曝出来的结果可信吗,会不会暗示蛋白表达量高,这样的蛋白需要减少上样量,把曝光时间控制在比如3min以内更合适呢,得到的结果更可信呢?求赐教
丁香园-
基础科研-蛋白质和糖学-
2018-09-22 14:18:04
wb封闭以后忘记洗膜了
wb封闭完之后忘记洗膜了,第一次做wb就这里出问题了,直接放在抗体里面了!这样对结果会有影响吗?怎样的影响,影响大不大啊?真的好伤心,听师兄说这里很关键,要洗四次。上网查有很多都说不用洗。有大神回答
丁香园-
基础科研-细胞生物与生物信息-
2022-07-28 11:06:56
WB封闭时间两个小时,有什么影响
我今天在做WB时,记错了时间,5%脱脂牛奶封闭了两个小时,请问各位,有什么影响
丁香园-
基础科研-蛋白质和糖学-
2019-10-26 14:37:37
请问一下师兄师姐wb目的蛋白可以在不同时间段收吗
rt。大部分蛋白我都是24小时收的,因为这个点造模组变化相对比较明显。但是做了时间梯度发现 有一种A蛋白它的变化趋势是6小时最低,24小时时趋向于恢复至对照,那么我可以在lps刺激6h后单独收
丁香园-
基础科研-蛋白质和糖学-
2023-04-15 17:18:12
有关WB中的封闭液存放时间问题
我封闭液和抗体的配置,都是用脱脂奶粉,但是中间几天没做,见天做时发现,液奶粉上面是绿色的液体,不知道能不能用,一般,配置的封闭液和抗体,可放多久?
丁香园-
基础科研-实验动物与生化组胚-
2016-05-09 13:15:27
Wb经验帖‖时间等问题
又是科室抽空来骗赞的一天,昨天有人问wb时间问题,我做一个总结吧。1、分离胶20min±5min凝,冬天偏短。2、浓缩胶压10min钟,压完1h在4°冷藏后再用3:电泳时间不固定,不固定,不固定
丁香园-
基础科研-蛋白质和糖学-
2022-07-28 02:28:54
【求助】WB的封闭问题
问大家一个比较菜的问题,做WB封闭时,我用脱脂奶粉封闭后也用TBST洗了三次,因为感觉脱脂奶粉会不会影响后面一抗的孵育,后面看资料没提到要洗,有的还说一抗用封闭液稀释,一抗用脱脂奶粉稀释没问题
丁香园-
基础科研-蛋白质和糖学-
2010-05-20 10:06:42
WB没封闭直接孵了一抗影响大吗
分子量200+的目标蛋白,我给搞忘记封闭了,可不可以用TBST多洗几次以后孵了二抗去显影真的不想重新来了
丁香园-
基础科研-细胞生物与生物信息-
2023-03-01 03:26:44
【求助】关于WB封闭过夜的问题
我做WB转完膜后用5%的脱脂奶封闭过夜,我想请教各位大虾,用BSA做封闭液和脱脂奶有什么不同吗?二者哪个更好些?
丁香园-
基础科研-蛋白质和糖学-
2006-06-11 07:05:23
Wb‖忘记封闭了,直接加了一抗怎么处理?
了牛奶封闭杂位点,物理性一抗结合增多,所以会多余展示结果,假阳性,tbst洗脱就会除去这个物理性结合,但是tbst洗的时间长了,会洗脱目的蛋白,所以会有点浅!
丁香园-
基础科研-蛋白质和糖学-
2022-04-25 10:55:10
【求助】【求教】WB中有关TBST和封闭的问题
各位大侠,小弟最近在研究有关WB的理论问题,但是有一事不明,希望得到赐教: 转膜后的封闭理论上来说是为了阻断可以和一抗二抗非特异性结合的位点,从而使一抗二抗可以充分进行特异性结合,但是在TBST
丁香园-
基础科研-蛋白质和糖学-
2012-08-20 09:02:44
求助!蛋白大小在7kda左右做WB的转膜电压还有时间应该在多少啊
求助!蛋白大小在7kda左右做WB的转膜电压还有时间应该在多少啊
丁香园-
学习交流互助专区-
2023-06-30 12:50:12
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western blot 操作步骤详解
2016-08-11 12:36
来源:生物学霸
作者:dlzhangyu
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上回说到如何进行蛋白质的样品制备、蛋白质定量及 SDS-PAGE 电泳,如果你还没看过,点此详见:《献给初学者:western blot 操作步骤详解》。下面就跟大家讲讲接下来的步骤。转膜我们实验室使用的是半干转膜电泳槽。对于转印 90 kd 以下的蛋白,转膜液都不用加 SDS, 如果是蛋白分子量大的话可以加入 0.037% 的 SDS。1. 在电泳结束前 20 min 开始准备转膜所需的东西。转一张膜需准备 6 张的滤纸(长一般为 8.1~8.3 cm,宽度根据裁的胶大小实际测量,但胶一般会缩水,所以裁 8 cm 就行)和 1 张 PVDF 膜。切滤纸和膜时一定要戴干净手套,因为手上的蛋白会污染膜。PVDF 膜使用前用无水甲醇中浸泡 1 min~2 min。目的是为了活化 PVDF 膜上面的正电基团,使它更容易跟带负电的蛋白质结合,做小分子的蛋白转移时多加甲醇也是这个目的。2. 将玻璃板撬开才可剥胶,撬的时候动作要轻,要在两个边上轻轻的反复撬。撬一会儿玻璃板便开始松动,直到撬去玻板(撬时一定要小心,玻板很脆弱,我用的是冰箱里附带的塑料除霜铲,效果出奇的好)。除去小玻璃板后,将浓缩胶轻轻刮去,要避免把分离胶刮破。也可取 10 ml 注射器吸满转印缓冲液,往玻璃板与凝胶之间注水,使液体的压力将两者自然分开。取出凝胶后应注意分清上下,可以在右上角裁一个小角。之后有两种方法供选择,一是按照 marker 指示,把含有自己感兴趣的蛋白的胶裁下来,二是把整张胶转膜,前一种方法更加节约材料,但操作稍微麻烦了一点。在加有转移液的培养皿里放入裁好的胶、浸过甲醇的 PVDF 膜和滤纸,平衡 10 min 左右,也是为了除去滤纸和转移膜中的气泡以及胶上多余的 SDS。3. 带上手套,平放转移槽的底座,依次在石墨电极上叠放 3 张浸泡过缓冲液的滤纸、PVDF 膜(此时可在 PVDF 右上角减去一个角,以辨明转膜后的蛋白面与非蛋白面)、刚刚完成电泳的凝胶和另外 3 张浸泡过缓冲液的滤纸。用枪头或移液管在叠层的滤纸上滚动,除去气泡。注意每叠一层就要用玻璃棒或圆筒试管赶去气泡,因为一个气泡的厚度对于蛋白来说都是十万八千里的。用干净纱布将叠层上面和周围的多余缓冲液吸干(这一步很重要,宁可太干也不能太湿,太干容易烧胡,太湿的话多余的缓冲液会导致电流短路,大大降低转移效率,如果同时转好几条胶的时候更要小心,有一个胶短路就会影响整体的转移效率)。最后将转移槽的上盖扣上,接通电源开始转膜。由于设备昂贵,第一次操作应向熟手请教,否则短路可能烧坏设备!转完后立即清洗设备,特别是金属上盖,转膜液特别容易在金属上盖上形成结痂,影响设备的正常使用,我们实验室今年做 western 的同志因为忽略这一点,转膜仪烧坏了好几次。4. 依据分子量大小电泳 15~60 min。如果初次转膜,可以根据一般经验,即多少 kD 的蛋白就转多少分钟,再根据结果调整时间。之后断开电源,取出转移膜进行后继实验。5. 为了便于观察电泳效果和转膜效果,以及判断蛋白分子量大小,最好使用预染。传统方法是将膜用 1×丽春红染液染 5 min(于脱色摇床上摇)。然后用水冲洗掉没染上的染液就可看到膜上的蛋白。实际上更常用且简便的方法是先将膜晾干,然后浸入 20% 的甲醇,待完全浸湿后取出透光观察即可看到蛋白条带(此方法仅仅适合 PVDF 膜)。将膜晾干备用。使用预染 marker 话可以看见 marker 的颜色转印到了膜上,但是凭借这个颜色来判断是否转印完全或转印过头是不可靠的。免疫杂交反应1. 将膜移至含有封闭液的平皿中,室温下脱色摇床上摇动封闭 1 h 即可。如果是自己配置的封闭液,最好过滤一下以消除固体杂质。实际经验表明与其封闭过夜,不如一抗孵育过夜。2. 将一抗用封闭液稀释(一般用封闭液稀释即可,如果背景不高用 TBST 稀释也可以,当然熟练后还可以自由发挥,配制一些自己的复方稀释液)至适当浓度(可以在 1.5 mlEP 管中配置工作液,常用稀释倍数为 1:200,1:500,1:1 000,具体需要预实验进行摸索,用后可回收重复用 2~3 次,但回收重复利用的效果如何就要看抗体的质量,分装的抗体不推荐回收。稀释后应在 2-3 天内使用,4 度保存,避免反复冻融。)撕下适当大小的一块儿保鲜膜铺于实验台面上,四角用水浸湿以使保鲜膜保持平整;将抗体溶液加到保鲜膜上;从封闭液中取出膜,用滤纸吸去残留液后,将膜蛋白面朝下放于抗体液面上,掀动膜四角以赶出残留气泡(也可使用手术室的病理袋,质量不错,减去下面的折叠部分,用封口器(40~80 元一个)封上,不漏液即可)。37° 孵育 1 h 之后转移到室温下再孵育 1 h(或者 4°孵育过夜),用 TBST 在室温下脱色摇床上洗两次,每次 10 min;再用 TBS 洗一次,10 min。也可以多洗几次,比如 4 次,每次 5 min。3. 在培养皿内加入二抗稀释液(10 ml 足够了,一般 1:1 000 甚至 1:10 000 用 TBST 稀释),放在摇床上,室温下孵育 1~2 h 后,用 TBST 在室温下脱色摇床上洗两次,每次 10 min;再用 TBS 洗一次,10 min, 进行化学发光反应。选择好一个合适的条件不要轻易改变,另外相比一抗,二抗作用的时间应严格控制,实践证明一抗时间长点可能没什么关系,而二抗时间若过长过短都将会直接影响结果。二抗孵育之后还要注意好好洗涤,否则显影是背景可能脏。化学发光(ECL),显影,定影1. 现在工作台上铺一张保鲜膜,将 PVDF 膜放在保鲜膜上。将 ECL 的 A 和 B 两种试剂在 EP 管内等体积混合(注意吸完 A 试剂后吸 B 试剂前要患枪头),然后均匀滴在 PVDF 膜的蛋白面,反应 1~2 min 后,将 PVDF 膜上多余的 ECL 工作液吸干,之后转移到在 X 片夹中预先铺好的保鲜膜上一侧,把另一侧翻过来盖在其上。用透明胶把保鲜膜固定在片夹上。2. 在暗室中,将 1×显影液和定影液分别到入塑料盘中;在红灯下取出 X-光片,用切纸刀剪裁适当大小(比膜的长和宽均需大 1 cm);打开 X-光片夹,把 X-光片放在膜上,一旦放上,便不能移动,关上 X-光片夹,开始计时;根据信号的强弱适当调整曝光时间,一般为 1 min 到 2 min,也可选择不同时间多次压片,以达最佳效果(也有人重叠压好几张片,固定曝光 5~10 分钟,从这好几张片中选出最合适的底片);曝光完成后,打开 X-光片夹,取出 X-光片,迅速浸入显影液中显影,待出现明显条带后,即刻终止显影。显影时间一般为 1~2 min(20~25℃),温度过低时(低于 16℃)需适当延长显影时间;显影结束后,马上把 X-光片浸入定影液中,定影时间一般为 5~10 min, 以胶片透明为止;用自来水冲去残留的定影液后,室温下晾干(注意:显影和定影需移动胶片时,尽量拿胶片一角,手指甲不要划伤胶片,否则会对结果产生影响)。X 光片一般选用柯达原装的生物实验专用柯达 X-OMATBT 胶片 . 显影液和定影液最好每周配置一次,避光室温保存,显影和定影液是最便宜的试剂了,千万不要因为它而影响了整个结果。凝胶图象分析将胶片进行扫描或拍照,用凝胶图象处理系统分析目标带的分子量和净光密度值,比如 bio-rad 的 Quantity One。如何使用敬请关注我们下一期内容。主要参考资料WB 实战指南+正确使用 Quantity One 定量 by jacqueslm2001Abcam 的 western 新手指南土豆网的 western blot 视频 上海交大出品《分子克隆实验指南》精编版 化学工业出版社《抗体技术实验指南》科学技术出版社 milipore 的 western blot 的优化方案Western blot 步步看--网络流传最广的资料,作者不详Bio-rad 蛋白印迹手册穷人的劳斯莱斯-我的五年 western blot 体会附:Western Blot Quick Guide 以及一天跑完 Western 的时间规划1. 前期准备:蛋白已经定量,各样本已经稀释到某固定浓度,已经和 SDS loading buffer 混合,已经分装好,保存与 -20°。头天下午或晚上配胶,分离胶和浓缩胶一起配好,带上梳子,放入潮湿的自封袋内放入 4° 冰箱备用。2. 8:00 从冰箱里取出蛋白样品,用 PCR 仪 95 度加热 5 min。混合均匀并离心。3. 8:30 取出昨晚配好的胶,组合,加电泳液。在电泳液里拔梳子能稍微容易一些。用 10 uL 的枪加样。4. 9:00 开始电泳。恒流 30 mA 一般 1 个小时足够了。5. 9:30 开始准备转膜用的滤纸和 PVDF 膜,PVDF 膜泡甲醇。一般 8 cm 的宽是固定的,所以如果要裁胶的话可以先大概估计一下。6. 10:10 电泳结束(假设电泳用了 70 min),起开玻璃板,裁出胶上所要的条带,如果整块胶转就更方便了。把胶放在盛有转膜缓冲液的培养皿里。7. 11:00 转膜开始(这里裁纸和铺三明治还是比较费时间的,我一般要用 30 ~ 50 min,所以这里要抓紧时间)。8. 12:00 转膜结束(这里按 60 min 的半干转的时间来计算)。开始封闭 1 h。9. 13:00 封闭结束,开始孵育一抗(在这里你可以选择一抗孵育过夜,如果不过夜的那一天就能结束),如果不过夜的话我一般选择 37° 1 h 然后在室温(23°左右)再 1 h。10. 15:00 一抗孵育结束。TBST 洗涤 3*10 min 或者 5*6 min。11. 15:30 开始孵育二抗。室温 1 h 足够了。12. 16:30 二抗孵育结束。TBST 洗涤 3*10 min 或者 5*6 min,这里推荐采用 5*6 min。13. 17:00 ECL 发光,压片 10 min,显影 1 min,定影 10 min,那么在 18:00 之前你就能看到结果。如果结果不好还有后招,用 stripping(一抗二抗洗脱液)洗涤,我用的是碧云天的碱性一抗二抗洗脱液,按照说明书来一般 20 min 就行,然后洗涤几次,重新封闭 1 h,然后一抗过夜,明日再继续战斗。这样的话 18:30 之前也能结束战斗。文章来源:丁香园站友 @dlzhangyu
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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
Sign Up for Western Blotting Tips and Webinar Alerts
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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Western Blot University
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蛋白质印迹(Western blot)实验方案
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荧光 WB 指南
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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首页 实验方法 细胞技术 细胞功能测定 Western Blot实验中转膜和封闭的注意事项
Western Blot实验中转膜和封闭的注意事项
Western Blot实验中转膜和封闭的注意事项
关键词: 实验 转膜
来源: 互联网
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相关专题
生物实验中的封闭剂
经典的转膜方法是电转移,因为电转移需要配套垂直电泳槽 来进行。如何做这个“海绵—几层滤纸—胶—膜—几层滤纸—海绵”的“三文治夹心”,在分子克隆上已经有详细的介绍,相信大家都很容易找到。一些细节:
记得全程手套操作,一则避免手印污染影响结果,二则保护自己(未交联的丙烯酰胺、甲醛等等虽不立即致命但都会慢慢毒害你的身体,可不要这样白白为科学献身哈)
如果WB前没有可参考的资料——比如不知道是否有表达,比如抗体少还要摸条件(稀释度),可以用剪膜剩下的边角料来先做几组点杂交摸条件,省点时间省点试剂;
去掉积层胶后,预染的Marker可用以识别胶上下方向和膜的正反面(预染Marker如果照着说明书用量,有可能在电泳时看不到条带,但转膜时有浓缩效应而且背景白就可以看到了。如果要电泳能看到就要参考电泳的那个用量,或者多加1—2倍的量);如果目标蛋白小,指示剂也可以指示方向,但是如果蛋白大,指示剂已经跑出去了,就要留意分清胶上下方向,切个小角是常用的方法。膜上要做好标记,识别正反面和上下。剪一个小小角最方便。膜和滤纸一起裁最好(不过滤纸上下叠多了膜不好剪,硝纤膜容易裂,用利刀+尺子+垫厚报纸划比较容易)尽量和胶一样大小,胶用纯水冲洗一下后用电泳缓冲液平衡过再量。我自己通常剪的时候会故意长宽各比胶少1mm,保证膜和胶不会碰到对方背后的滤纸就好。
对于特别小的蛋白,tricine SDS-PAGE电泳 有助于提高蛋白大小在1KD—20KD间的分辨率,不用甘氨酸,丙烯酰胺的浓度也不用太高(可参考2004年中国生物工程杂志上有一篇文章(有效分离1kD小肽的Tricine-SDS-PAGE方法),87年Anal.Biochem也有一篇文献)。
转膜前胶要在转移缓冲液里平衡一下防止胶变形,也有助于进一步去掉可能有碍转膜的杂质。还有人在这一步浸泡帮助蛋白复性,可以直接在胶上检测蛋白活性。
膜漂在水面(或者甲醇液面)让液体从下通过膜上的孔渗上来以赶走膜内空气,膜彻底浸润后颜色会变深一点,任何白点或者斑都是没有完全浸润的标志,会影响转膜的。最后浸没入缓冲液里平衡。甲醇处理PVDF不要超过15秒。以后的步骤中不要让膜干涸了,万一不幸发生也用同样处理。
电转移缓冲液通常用Tris glycine系统,如果是转膜后有部分样品要蛋白测序 ,最好用CAPS缓冲液,减少甘氨酸对测序的污染。
半干电转移(Semi-Dry)用经过缓冲液饱和的滤纸代替传统转移槽,非常节约试剂,而且效果也好。由于不用“泡”在缓冲液中,半干转不单可用均一缓冲体系,也可以做非均一转移缓冲体系(配方下面有,还可以加 20 % (v/v) 甲醇,据说有助于增加转膜效率和减少胶变形的,但据说也有可能影响抗体识别)。半干转可以在30分钟内完成转膜(每平方厘米电流2.5—3.5mA,恒流,冷库。如果电流要求小可以延长到60—90分钟,但要防止过热。如果有那种温度贴,可以贴上参考温度),即使205KD这么大的蛋白转膜效率也高达80%。各种大小的蛋白的转移效率都OK。半干转可以上下层叠2块胶+膜一起转(面积还是按照单个计算),或者并排放(2块胶的面积计算),只要控制好单位面积的电流强度和时间,防止过热就好了。
Discontinuous blotting buffer to be used:
Anode buffer I:
300 mM Tris
Anode buffer II:
30 mM Tris
Cathode buffer:
25 mM Tris/HCl (pH 9,4) 40 mM 6-Aminocapronic acid
有人觉得转膜加SDS有助于大分子 蛋白转膜,我个人持保留意见,因为SDS等去垢剂影响硝纤膜和蛋白的结合,反而不好。
如果只做western blot ,膜可以用丽春红S染色并在脱色前照相,对后面的免疫反应影响不大。不要用考马斯亮蓝或者氨基黑染色膜以免影响结果。如果有预染Marker需要用针或者笔扎眼记录条带位置,以免后面会洗掉而无法判断结果。预染Marker也有助于判断转膜的效率和情况,如果比目标分子大的Marker都已经转过去了,那就OK了。如果有能在Western结果显色的Marker(比如生物通前面特别推荐的别出心裁 特别好用的蛋白分子量标准 谁可小看“蛋白标准”(下) )就更方便了。
有人说在中性条件下电泳有助于蛋白测序,如果要蛋白测序需要提前一天倒胶,并说预电泳6小时(有还原剂如Glycolate)后再倒积层胶。除了要做HPLC分析应该用丽春红S而不要用考马斯亮蓝或者氨基黑染色,其他的比如测序和或者PTH都可以选灵敏度高些的考马斯亮蓝或者氨基黑。没条件做,参考而已。
转印后的PVDF膜在含20%甲醇溶液中在白透射光照下不用染色也依稀可以看到透明的蛋白条带,有时这种快速的方法可以不用染色识别条带,避免染色膜影响后继实验。
转膜后的封闭注意:
转膜后,膜上其他的空白位置需要用封闭液封闭。Western的灵敏度某种程度上受限于封闭做的好不好。脱脂奶是最常用的经济配方(实验太晚了还可以补充一下营养,哈哈),用这种封闭剂由于里面可能有痕量的生物素和碱性磷酸酶,可能造成背景污染而不适合生物素—亲和素的检测方法(如果用了生物素标记的Marker而且结果背景较高,分析结果也有可能是受此影响),脱脂奶也不适合碱性磷酸酶检测(AP)方法。
如果采用碱性磷酸酶检测系统,封闭剂最好用6%酪蛋白+1%聚乙烯吡咯烷酮+10mmol/L EDTA磷酸缓冲盐加热65度1小时确保碱性磷酸酶失活(可以加0.05%叠氮化钠,新鲜最好)。经济的脱脂奶配方和AP兼容得不太好,再加上HRP的底物选择范围也更宽,现在HRP是越来越普遍的选择。但是叠氮钠(NaN3)对辣根过氮化物酶(HRP)有灭活作用,如果用HRP检测系统则封闭液不要加叠氮化钠为好。切记。封闭时间和封闭剂的量都要足够。封闭不完全,后面就全白忙活了。
如果选用AP作为显色方法,封闭时就要选择Tris缓冲体系,不要用PBS,因为PBS干扰AP。再想起什么再补充吧。下一篇转到显色试剂的选择了.
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