i***0
发帖数: 160 | 1 HI,I have a question about Western Blot. We have immunized rabbit and
obtained polyclonal antibody. using the purified antibody from rabbit serum,
we could see specific band toward our antigen. However, with ECL
luminescence, I always have a dirty background, with dots and cloudy smear
that make the image so ugly and not publishable. Do you have any suggestions
? I appreciate a lot for your help! | i***0
发帖数: 160 | 2 I have tried increase wash volume, wash time, times of wash, adjusting
antibody concentration. Occasionally, I got a relatively clean image, but
still not clean. Someone told me I could try add some salt in PBST for wash,
and I am trying it today. I will report my result when I got it. But if you
have any great tricks, please help. Thanks | h*******o
发帖数: 4884 | 3 1) try other blocking solution and block longer
try BSA, non-fat milk, or both and block at least for 1 hour
2) Reduce your 2nd antibody concentration.
3) For primary antibody incubation, incubate in blocking buffer and try
either 1-2 hr at RT or 4oc overnight, whichever one you haven't tried yet | i***0
发帖数: 160 | 4 Thank you very much! Currently I am using 5% milk and 10^4 times of dilution
for secondary antibody.But I will try BSA next time. I have tried primary 1
hr at RT and O/N, and it seems better with primary 1 hr. I just hate
trouble shooting. | i***0
发帖数: 160 | 5 It turns out great with 100 mM NaCl PBST washing. In addition to that I
increase wash times from 3 to 5, and rinse with fresh PBST before put the
membrane in PBST wash box. |
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