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I see bands with western blot only the third day!


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#1 chemokeen

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Posted 12 November 2010 - 11:00 AM

Hi, i am worried lately because of my results at western blot and any help could be more than welcome. I am using the classic procedure. First day, electrophoresis of my samples (human serum), transfer at NC or PVDF (i got the same results), blocking for 1h with 5% non-fat milk, washes with PBST incubation overnight at 4 C with 1st Ab. At second day, I begin with washes with PBST and then incubate 2nd Ab at room temperature, then washes again and finally darkroom. I get nothing.. A clear film. The first day i watched this i was worried that maybe the 1st or the 2nd Ab didn't bind at all, so i took the membrane and without stripping i repeated all the stages of protocol after blocking (blocking for 30 min and then the same). The only thing that is altered is the incubation of 1st Ab. I incubated it for 1h at RT. I got some bands then very faint close to the MW i expected. I repeated the experiment for other 2 times and it gave me the same result. Results only at third day. Is this right? So what to assume? Is there a possibility the 1st Ab binding non-specifically at RT due to higher Kaffinity? Is there a possibility the 2nd Ab binding at membrane? I am currently working on the latter without incubation of the 1st Ab. Any suggestions? Thank you. :huh:

#2 madrius1

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Posted 12 November 2010 - 11:52 AM

Have you tried a positive control in your blot? Has this antibody ever worked in your (or others) hands? Are you sure the transfert is effective (even though it seems like it, since you can see MW)?

Incubating ar RT will help if your Ab binds very weakly to your protein of interest. You could try reducing the washing time, increasing the amount of protein you load or try another antibody. Some secundary abs sometimes bind to membrane/other proteins. But as you describe it, it seems to me that the problem is your primary antibody.

#3 chemokeen

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Posted 12 November 2010 - 12:07 PM

Have you tried a positive control in your blot? Has this antibody ever worked in your (or others) hands? Are you sure the transfert is effective (even though it seems like it, since you can see MW)?

Incubating ar RT will help if your Ab binds very weakly to your protein of interest. You could try reducing the washing time, increasing the amount of protein you load or try another antibody. Some secundary abs sometimes bind to membrane/other proteins. But as you describe it, it seems to me that the problem is your primary antibody.


Actually i haven't used positive control because i have to use extracts from tissue that give positive results. Transfer conditions seem to be ok 1h at 200 mA and another h at 250 mA. I see the proteins from the ladder at membrane, so i think it's ok. The first Ab has been used in my lab so i don't think that's the case. Also the manufacturer is reliable. I usually store Abs in order to re-use them. I store them at 1% non-fat milk and 0.02% NaN3 (the latter only for 1st Abs). Do you think may this be a problem? Should i try BSA instead of milk? I will try decrease washing.. Thank you for your reply.




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