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- Proteins and Peptides
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1.Problem: The attachment is the blot result of Hela cell lysates using antibody(ab791), no band could be detected even exposure 60min(A). the same membrane was reblotted by anti-Arp3 antibody, obvious band could be found(exposure 1min, B).It look to me that antibody(ab791) of your company does not work at all. So I'd like to have credit back. 2.Protocol: Hela cells were grown to 90% confluence in 100mm dish, washed once with 1(JPBS, then add 200(Jl SDS protein sample buffer, saved cell sample and boiled at 95(JC for 10min. 20(Jl sample were loaded and separated by 10% SDS-PAGE and then immunoblotted with antibody-BAI1 associated protein 2 (80(Jl ab791 was added into 8ml milk, incubate at 4(JC overnight). The membrane was washed three time with 1* TBST(pH 7.4), then add stabilized goat anti-Rabbit HRP-conjugated(1:10000, incubate 60min at RT). Blot were visualized with enhanced chemiluminescence(ECL; Amersham Pharmacia Biotech) and autoradiography. The same blot was stripped and reblotted with Arp3 antibody to confirm the expression of Arp3 in total cell lysates of Hela cells. Secondly antibody both used were the same, So I can make sure the problem was not come from the secondary antibody. You can mail me if you have any question about it. Thank you. |
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ANSWER: |
Thank you for your enquiry. AB791 is "Goat polyclonal to BAI1 associated protein 2 isoform 3" and as we understand from your e-mail you are using Goat anti-Rabbit secondary. There seems to be some problem with the compatibility of the primary and the secondary antibody. We would suggest using either Donkey or Rabbit polyclonal to Goat IgG. |
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