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wdwd123铁杆木虫 (职业作家)
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[交流]
PVDF膜的本底问题
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各位大哥大姐，谁用过PVDF膜呀？我做的是抗体膜芯片，我用5%的脱脂牛奶封闭，常温下2-3个小时或4度过夜，还有很高的本底。 大家做western-blotting的时候也用PVDF膜呀，好像文献上说封闭效果都很好啊，为什么我那样做就不行呢？ 我是这样做的，先用5%牛奶封闭，然后点二抗上去，TBST洗后，再加底物，结果却显色了，按理说，不该显色啊。 等等，中间有很多问题，希望大家给点建议。 如果有同行，我们交流下：506932097 也希望做western的朋友们给点建议。 [ Last edited by 350784478 on 2009-10-2 at 13:43 ] |
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sutao1979
木虫 (著名写手)
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wdwd123(金币+8,VIP+0):谢谢 4-25 19:54
wdwd123(金币+8,VIP+0):谢谢 4-25 19:54
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以下是一些trouble shooting，问题的解决办法，可以参考 1Blocking of non-specific binding might be absent or insufficient. Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5% non-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well. 2The primary antibody concentration may be too high. Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best). 3Incubation temperature may be too high. Incubate blot at 4°C. 4The secondary antibody may be binding non-specifically or reacting with the blocking reagent. Run a secondary control without primary antibody. 5Cross-reaction between blocking agent and primary or secondary. Add a mild detergent such as Tween20 to the incubation and washing buffer. 6(phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Use BSA as a blocking reagent instead of milk. 7Washing of unbound antibodies may be insufficient. Increase the number of washes. 8Your choice of membrane may give high background. Nitrocellulose membrane is considered to give less background than PVDF. 9The membrane has dried out. Care should be taken to prevent the membrane from drying out during incubation. [ Last edited by sutao1979 on 2009-4-26 at 18:54 ] |
4楼2009-04-25 18:59:53
sutao1979
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2楼2009-04-25 15:51:25
wdwd123
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3楼2009-04-25 17:03:58
winter_gates
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5楼2009-04-26 10:40:05
