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Cause of cloudy Western blots?


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#1 SarahC

SarahC

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Posted 04 May 2011 - 10:21 AM

I have been using streptavidin-conjugated Dynabeads to pull biotinylated cell surface proteins out of whole cell lysate. After eluting the proteins I run them on a 4-12% Bis-Tris gel and transfer to nitrocellulose using an iBlot machine. I have been probing for a nAChR subunit with a polyclonal antibody, and all of my blots looked cloudy and smeared over the entire membrane. I did multiple experiments to test whether the problem was coming from my blocking buffer (5% BSA in PBST) or my elution buffer (6M urea with LDS). I tried milk for blocking, not heating the sample during the elution step, and using a glycine elution buffer instead, and all the blots were still cloudy. Now I have run one where I used the same elution and blocking conditions for both pieces of membrane but used the polyclonal antibody on one and a monoclonal on the other, and the one with the monoclonal looked fine. However, my coworker is adamant that there is no reason for the antibody to be causing this and says that he has used the same antibody and gotten good results (he does not use biotin/streptavidin or the urea elution buffer). Is there any way my conditions could be interacting strangely with the antibody, or is there another reason I have not thought of, or a good explanation I can give him? Thank you for any help :)




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