Western&blot发光检测中常见的问题及解决方法
免疫印迹试验（Western blot）是分子生物学、生物化学和免疫遗传学中常用的一种实验方法，被广泛应用于蛋白表达水平的研究、抗体活性检测和早期疾病诊断等多个方面。其中较为简单也为大家所普遍接受的Western blot显色方法为底物化学发光法ECL，文章根据发光检测中常见的几种问题现象与各位生物人一起探讨。
众所周知，Western blot的原理是蛋白质在电力场的作用下，由大到小的进行排列，利用电泳进行分离和富集。拥有抗原表位的蛋白质分子被抗体（一抗）特异性识别并与之结合。在此基础上，酶或荧光标记的二抗识别并结合一抗，通过与底物反应显色来观察目的蛋白。
Western blot显色的方法主要有以下几种：一、放射自显影，二、 底物化学发光ECL，三、底物荧光ECF，四、底物DAB呈色。现常用的有底物化学发光ECL和底物DAB呈色。大部分Western blot显色底物是化学发光底物，发表文章通常也是用底物化学发光ECL。
ECL法检测辣根过氧化物酶的原理是辣根过氧化物酶在H2O2存在下，氧化化学发光物质鲁米诺（lumino，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，通过将印记放在照相底片上感光就可以检测辣根过氧化物酶的存在。ECL法操作简便，应用现成的试剂盒，按照说明，将两种显色底物等体积混合后将其覆盖在膜表面使其均匀，用玻璃胶片把膜包起来，马上在暗室中将X光片覆盖在膜的上面，显影、定影（或用荧光检测仪检测）。ECL法具有灵敏度高，线性范围广等特点。
图1 ECL检测原理图
Western blot发光检测受多种因素影响，主要包括：抗原含量，抗体敏感度，底物敏感度，显影和定影效率，等。现根据检测中几种常见的现象问题进行分析：
1.目的蛋白信号弱或无。在Western blot发光检测中常见的问题之一就是目的蛋白信号弱或无。首先考虑是否转膜不充分/过转，如果是，则要优化转膜条件；其次，在转膜合适的前提下考虑是否抗体-抗原敏感性过低，也可能是底物HRP或底物活性降低，可以通过加大上样量/提高抗体浓度/使用敏感底物/延长压片时间解决。曝光时间短也会使蛋白信号降低，因此，要延长胶片的曝光时间。
2.背景较高。 背景深是在Western blot发光检测中最常见的问题，造成背景深的原因很多。第一，封闭的问题，封闭条件一般为5%牛奶或BAS室温封闭2-4h，但最好4度过夜；实际上牛奶对于多数抗体来说效果不错，但二抗与牛奶可能有交叉反应，因此洗涤很重要。另外BSA蛋白单一，可降低因牛奶其他成分引起的背景。第二，TBST洗脱不彻底也会导致背景深，适当增加洗脱时间、次数、用量可以减轻背景颜色。第三，条带背景深也与曝光时间有关，曝光时间超过半小时出现背景是很正常的，所以可以的话建议曝光1-10min。英格恩公司生产的Westernblot 发光试剂EnlightTM含独特的发光增强剂和精准的发光底物，比普通发光试剂灵敏度更高、更稳定，而且发光时间长，背景更低。第四，抗原-抗体浓度/HRP过高时，直接或间接结合在膜上，洗脱不掉等原因，考虑降低抗体浓度或蛋白上样量。
图2& Western blot发光检测中常见的问题及比较
3.非特异性条带。 非特异性条带与抗体交叉反应有关。单抗比多抗特异性好，纯度高，另外适当稀释一抗/二抗浓度，也是有必要的。如果样本在处理过程中发生降解，则样本处理时加入蛋白抑制剂在冰上操作。
4.显影后膜上条带处变为黄色或为白色。可能是HRP过高导致的敏感性或背景太高，应降低抗体浓度。
5.条带内无显影的白点与转膜和显影的操作有关。转膜前尽量将膜平衡，并注意胶和膜之间避免气泡，显影时膜与X光片间也应避免气泡。
6.散在小圆斑是牛奶封闭液中的杂质颗粒导致的，解决此类问题可提前配置好封闭液于4度保存，使用时勿混匀，仅用上层。
blot发光检测中常见问题及解决办法
信号弱或无
HRP过高引起底物迅速耗竭
稀释一抗/二抗
抗体-抗原系统敏感性过低
提高抗体浓度/蛋白上样量
转膜不充分/过转
优化转膜条件
HRP或底物活性降低
测试活性或选用敏感底物
HRP过高(背景较高)
稀释一抗/二抗
封闭时间不足
延长封闭时间4度过夜最好
抗体与封闭液有交叉
更换封闭液(如3%BSA的TBST)
TBST洗脱不足
增加洗脱时间、次数、用量
曝光时间过长
降低曝光时间
抗原-抗体浓度过高
降低抗体浓度或蛋白上样量
条带内无显影的白点
转膜不良（如有气泡在胶-膜之间)
膜平衡不均/油脂污染
按说明书平衡膜/戴手套用平头镊子持膜
膜与X光片间有气泡
显影前去除气泡
非特异性条带
抗体交叉反应
稀释一抗/二抗
散在小圆斑
封闭液有杂质颗粒
静置牛奶使大颗粒沉淀，仅用上层
反影(白色条带,黑色背景)
HRP过高(背景较高)
稀释一抗/二抗
显影后膜上条带处变为黄色
HRP过高(敏感性太高)
稀释一抗/二抗
底物化学发光ECL是Western blot最常用的显色方法之一，因此，熟悉并掌握其中最常出现的问题及解决办法是做好Western blot发光检测的基础。
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为什么我做的western blot背景特别高？
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这个帖子发布于13年零141天前，其中的信息可能已发生改变或有所发展。
我从去年开始做，开始一直很漂亮，几乎没有背景后来从12月到现在每次的背景都很厉害我是用甲醇预先泡膜5min,在转膜液里平衡20min封闭3h，一抗过夜，二抗1h，中间洗膜10min×3
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BLOCK 用什么？是不是东西失效了？一抗浓度有变过吗？如果是另一种抗体，降低一抗浓度。不知一抗过夜的温度你用了多少？
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丁香园荣誉版主
我认为做Western每一步都很关键，你的封闭液很重要（你用的是？），一抗在4℃、二抗常温孵育，孵育时间越长，灵敏度越高，特异性越差。
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彻底更换缓冲液，包括封闭液和洗涤液，时间长了，缓冲体系有所改变，对试验的重复性肯定有影响了
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封闭3小时？有这个必要吗？你用blotto封闭吗？你的电泳玻璃板也要洗干净，不然会有些背景杂带，尤其是做ECL更要小心。If you are not using anti-phosphotyrosine antibodies, you can block with BLOTTO. If you are using anti-phosphotyrosine antibodies, you have to use BSA because the background with BLOTTO is very high.
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PCRfan edited on
看看这个吧：Bart's Cookbook---------------------------------------------------------------------------------- Kamps's Western Blotting Protocol --------------------------------------------------------------------------------Procedure Recipes Comments -------------------------------------------------------------------------------- ECL or autoradiography?ECL is an appealing technique because it is quick and very sensitive and does not expose the investigator to radioactivity. The use of radioiodinated protein A to detect bound antibodies is however usually a superior technique.It is much more quantitative, at least if used in conjunction with a Phosphorimager.It has a lower background. There is less staining of abundant proteins in the sample, and for reasons that aren't clear, protein A shows much less binding to antibodies present in immunoprecipitates than anti-immunoglobulin antibodies. ECL analysis of immunoprecipitates using a second antibody is almost always blighted by strong staining of the antibody bands and by the staining of abundant proteins in the sample.Blots developed with iodinated protein A can be re-exposed to film to obtain an optimal image. Alternatively, the Phosphorimager scan can be adjusted to give clear definition of bands.It is almost as fast as ECL. Despite what was indicated in earlier versions of this protocol, identical incubation and washing intervals can be used. Additionally, a successful blot stained with fresh iodinated protein A can often be visualized in 60 minutes with the Phosphorimager, yielding a quantitative, manipulable image.--------------------------------------------------------------------------------
ProcedureThis procedure was originally optimized by Mark Kamps for analysis with anti-phosphotyrosine antibodies. It is however applicable to almost any form of western blotting with minor modifications. --------------------------------------------------------------------------------1) Run an SDS polyacrylamide gel as you would normally.Before the gel has finished running, be sure that you have started de-gassing the transfer buffer.Some people soak the gel in transfer buffer for 15 min before assembling the transfer apparatus. Recently, we have been setting up the transfers immediately and things seem to work fine.We usually use Immobilon membranes for westerns. Although there are suggestions that nitrocellulose may give a lower background than Immoblion in ECL, most people prefer Immobilon even for ECL.If you are using Immobilon, be sure to wet it first in methanol briefly and then wash away the methanol with transfer buffer.If you are using a nitrocellulose membrane, soak in transfer buffer prior to assembling the sandwich.
--------------------------------------------------------------------------------2a) Assemble the transfer folder described below in a large, baking dish containing enough transfer buffer so that all layers are submerged. The orientation of the electrodes is also demonstrated.--------------------------------------------------------------------------------(+) POSITIVE ELECTRODE (ANODE)******************* Plastic cassetteIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Scotch-Brite padsThree Sheets Whatman 3MM---------------------- Nitrocellulose or Immobilon Filter---------------------- Polyacrylamide gelThree Sheets Whatman 3MMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Scotch-Brite pads******************* Plastic cassette(-) NEGATIVE ELECTRODE (CATHODE)--------------------------------------------------------------------------------Avoid air bubbles between any of the layers.--------------------------------------------------------------------------------3) Place the gel/filter sandwich in the holder such that the proteins will migrate from the gel to the membrane, in the direction of the positive (red) electrode (anode).4) Transfer for 60 min at 60 Volts (or 3600 Volt X Min).Some very small proteins will pass through the filter if subjected to electrophoresis for this length of time. If that is the case, shorten the electrophoresis. Conversely, some very large proteins do not transfer efficiently under these conditions and their recovery can be increased by longer electrophoresis.5) Disassemble. At this point you can either rinse the membrane with rinse buffer (or water) and let it dry until you are ready to do the incubation with antibody, or proceed directly.--------------------------------------------------------------------------------To block nonspecific binding sites, incubate the membrane for 15 to 30 min in 3% BSA in rinse buffer . This should be filtered prior to use.In Mark Kamps's original procedure, he used 5% BSA and 1% ovalbumin. 3% BSA appears to work just as well. 1% BSA may also work satisfactorily, but you may be pushing your luck if you use it.You can reuse the BSA blocking buffer repeatedly.--------------------------------------------------------------------------------If you are not using anti-phosphotyrosine antibodies, you can block with BLOTTO. If you are using anti-phosphotyrosine antibodies, you have to use BSA because the background with BLOTTO is very high.--------------------------------------------------------------------------------If you are using ECL, OMIT the azide. It interferes with the chemiluminescent chemistry.--------------------------------------------------------------------------------6) Incubate the blot with the appropriately diluted antisera for 1 hr at room temperature in 3% BSA in rinse buffer. For affinity purified anti-phosphotyrosine antibodies, we use 2 micrograms/ml. The origninal lab protocol called for a 2 hr incubation, but that seems unnecessary.--------------------------------------------------------------------------------7) Wash the filter2 x 5 min with rinsing buffer1 x 5 min with NP40 bufferthen 2 x 5 min with rinsing buffer.With anti-phosphotyrosine antibodies, longer washes reduce the signal, shorter washes increase the background.--------------------------------------------------------------------------------8) Incubate the filter for 30 min with 125I-protein A in 3% BSA in rinse buffer (You can re-use your blocking buffer for this). We use ICN's 68038 (approx. 30mCi/mg specific activity). We use 12-14 microCi (approx. 10 microliters) 125I-protein A in a volume of 30-35 ml blocking buffer. We reuse this solution 3 times without noticing a reduction in signal (see #6 in Comments).9) Wash again as in step 7.Damp dry on Whatman 3MM, place on another sheet of paper, overlay with saran wrap and expose to film with a screen or use a phosphorimager.10) Protein bound to the filter can be stained using a solution of 1microliter/ml Pelican india ink, 0.2% Tween-20, in Tris-buffered saline from the shelf (Margie's). Stain for approximately 20 to 60 minutes. Rinse with water for 1 hr. Dry flat. Be careful. Buffers containing Tween 20 can reduce the binding of antiphosphotyrosine antibodies.-------------------------------------------------------------------------------- Recipes--------------------------------------------------------------------------------Transfer buffer:Glycine 57.6 gTris Base 12.0 gSDS 3.0 gMethanol 800 mlH2O to 4 liters&Na3VO4 370 mg(only for analysis of phosphotyrosine-containing proteins).This solution should be de-gassed !--------------------------------------------------------------------------------Rinse buffer:10 mM Tris-HCl, pH7.40.15 M NaCl0.01% NaN3--------------------------------------------------------------------------------NP40 Rinse Solution:Rinsing buffer plus 0.05% NP40-------------------------------------------------------------------------------- Comments1) Reducing the SDS concentration in the transfer buffer by more than 30% inhibits the transfer of some specific proteins from the gel to nitrocellulose.2) If you've never used an antibody for western blotting before, it is a good idea to dilute it, say 1:200, 1:500, and 1:1000, and compare the signals and backgrounds that you get .3) Blocking times can be abbreviated if the &BLOTTO& technique is used. This involves blocking with non-fat dry milk and Tween 20. Although this procedure works as well as or better than BSA for most antisera, it results in a much higher background for anti-phosphotyrosine antisera.4) Protein standards should be run on the same gel if molecular weights are to be determined. All except myosin will transfer and they can be detected by staining with India ink. Transferring the markers has the advantage that the markers themselves are then part of the membrane surface and therefore will shrink and expand along with the samples themselves. Pre-stained markers are particularly useful because they can help you cut your blot or gel and provide visual confirmation that you transferred your proteins in the right direction.5) Diluted antisera can be reused. We have sometimes re-used the anti-phosphotyrosine antibody 10 times. You can prolong the useful lifetime of the diluted antibodies if you minimize the loss of liquid during handling. A good way to do this is to keep the diluted antibody in the container in which you incubate the blots. This eliminates losses during pouring and pipeting the antibodies.125I protein A ca however, it should be used only on blots that have been exposed to the same antibody because of the possibility of cross contamination. (i.e. anti-vinculin antibodies that were released into the 125I protein A from the previous blot now binding to the vinculin band on the next blot analyzed with anti-Src antibodies.)--------------------------------------------------------------------------------If you want to refer to this technique, cite:Kamps MP and Sefton BM, (1988) Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305-315 . [Abstract of this article]
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you'd better check your primary antibody.Impurity and degeneration will lead to the higher background. Beside, ECL reagent is needed to clear up before using developer to detect. If available,can u attached your film .We may have a further disscuss on your problem .
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提高封闭液的浓度。一，二抗体1－2小时足够。（室温）。洗膜时间要提高到15min, 4次。可以提高到1％ toween.Nacl提高到0.5M （洗涤液）。这些都可以有效降低背景的。
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非常感谢大家！！！我的封闭液是TBS中加了0.1％TWEEN和5％的non－fat milk，只用一次就倒掉了 non－fat milk是bio-rad买的，应该没有问题封闭我以前用2小时，后来以为时间短没封好才用3小时，也曾4度封过夜一抗4度过夜抗体浓度没变过，用TBS中加了0.1％TWEEN和5％的BSABSA是sigma新买的，二抗常温孵育1小时
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提高封闭液的浓度（已经很高了）。一，二抗体1－2小时足够。（室温）。洗膜时间要提高到15min, 4次。可以提高到1％ toween.（提高10倍，可以吗？）Nacl提高到0.5M （洗涤液）。（我原来用的是0.0137M啊）这些都可以有效降低背景的。 （有依据吗？）
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同意上面的观点。如果封闭没问题，最有可能就是一抗的问题了。
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高盐适合降低背景，TOWEEN 的浓度可以调的，从0.01--1％，你的背景太高可以采取这个方法，还可以提高封闭液浓度到10％。 买国产的脱脂牛奶就可以了。你不相信，就去看看书，有专门的蛋白杂交书籍。
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为了去背景直接用TBS洗膜，可以不加 MILK 或是 TWEEN如果以前可以做得出，现在无论如何背景都高，不如再查一下你用的TWEEN 是否出问题了。
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关于降低WB背景一般的策略BLOCK：最重要。可以用一个小的膜做平行的阴性对照，也可用其他试剂如BSA封闭。洗涤液： Nacl提高到0.5M （洗涤液）；可以提高到1％ tween；可单独的加一步NP40 buffer洗，或延长洗膜时间。总之，时间长，背景低，信号弱。抗体：杂质, 降解，污染都不行；孵育时间越长，特异性越差。可适当减少其作用的时间和浓度。 当然，试验过程中，所用的东西的洁净程度，包括电泳玻璃板也要洗干净，不然也会有些背景杂带。
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除了大家提到的可能原因之外,我觉得其实膜也应该考虑一下,你用的是NC膜还是PVDF膜,NC膜可能会污染油脂,这也是造成高背景的原因之一.不知您用膜也是用的和以前一样的吗?
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现在我改用含0.3％ tween-20,0.25M的NaCl的TBS-T洗膜15min×1后10min×3结果好很多
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现在我改用含0.3％ tween-20,0.25M的NaCl的TBS-T洗膜15min×1后10min×3结果好很多上面发错了！sorry！！
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贴张改良后的图两张：
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to anini 1.建议使用10%的脱脂奶粉PBS配置即可（一定要新鲜配置，重复用不要超过3次），将会大大减少背景。2.从你的的X光片上看，你的目的条带还是很清楚的，建议缩短曝光时间和显影时间（使用新的显影液）。
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背景感觉还是高了。显色时间长了。还有消弱的非特异带，降低显色时间可以解决。出现白色斑点应该是转膜时候有气泡。可以注意下。
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减少 二抗的时间，增加最后一次的洗膜时间和次数，应该会有帮助。
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