Anti-UBE3A antibody (ab3519)

?Response to your reply: 1, the samples were fresh prepared every time, without any freez/thawing cycle. 2, on the same membrane that abcam antibody detected about 60kDa bands, when using E6AP antibody from BD Pharmingen(cat No is 611417) can detect 100kDa band. I have attched the result in first email about this complaint, I can attch it again with this email. As your explaining, the E6AP in the sample degradated, How to explain that BD antibody can detect 100kDa band while abcam's just can detect 60kDa band? 3, the dilution recommendated by your datasheet is 1/2000. and the customer has tried 1/1000, but the result was the same. So I don't think that the concertration of primary antibody is useful for this problem.

ANSWER:

Thank you for your email. I have checked with the originator of this antibody and it does seem that the protein that was used was most likely degraded (when E6-AP degrades a 50 kDa band seen in Western blots). However, we really don't know why the other antibody is working and this one is not. Would you be able to send a Western blot image as a jpg attachment so we can review it? It would be very helpful. Thanks again.

??the additional reply to your questions: 1?On what material?whole cell lysate or other? are you testing the antibody in WB? whole cell extract of both Hela and HepG2 2, How did you prepare the lysate for the analysis (protease inhibitors etc)? Question: Could you please confirm if you have used RIPA buffer for preparing cell lysate. If not then we would strongly suggest using it. The lysis buffer the customer used is universal buffer for IP , according to Journal of biological chemistry , vol 279 NO,11 pp,10167-10175. It's not a RIPA buffer. But we don't think that the buffer is the key reason, for using another E6AP antibody from BD Pharmingen on the same membrane, he can got right band(100kDa). I have sent the picture to you with my first 3?How much protein did you load? 40ul whole cell extract of Hela 4?At what dilution(s) have you tested this antibody? the customer has tried 1?2000 and 1?1000 dilution

ANSWER:

Thank you for the details that you have provided. Please note that E6-AP is a very labile protein. Freeze/thaw cycling of samples results in E6-AP degradation which is observed as a prominent 50 kDa band seen in Western blots. This may explain why your customer is seeing a band at approximately 60 kDa. So we suggest running fresh samples, and in addition, the concentration of antibody and incubation period may need to be increased. Please let us know if you need additional assistance.