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Anti-VAV3 antibody [EP1130Y] (ab52938)

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1 question for ab52938

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Question 1

Tuesday 03-January-2012

I loaded 20 ug of whole cell lysate and ran an SDS-PAGE. After transfer, the PVDF was blocked for 1 hour in milk and then incubated with primary antibody. The first antibody that I purchased was tried at various dilutions from 1:300 to 1:10,000, all in milk for one hour at room temperature. Secondary antibody was 1:10,000 for one hour at room temperature. I have tried the second antibody at 1:500 and 1:1000, both in milk. There are >10 bands on a 10% gel on the shortest exposure. I have tried both Hela cells and a bronchial epithelial cell line. The dominant bands are doublets at 56 and 34 kDa. I transfected these cells with siRNAs to Vav3 and see no decrease in levels in any of the bands compared to control with the exception of one band at ˜32 kDa. Any explanation for these results? Thanks for your help

ANSWER:

  

Thanks for the reply. I am a little stumped as to why you are having these issues. I can only think of 2 things that may help (and I am sure you have already tried them): 1 - One reason for multiply bands is that there is not enough protease inhibitor in your sample buffer. Increasing the concentration of this would help prevent breakdown of your protein. 2 - Switching to a 5% BSA block would also help reduce the non-specific binding that is occurring. Other than that, I honestly cannot think what else you could try. If you have already tried the above variations and they did not prove successful then I would be happy to either replace this antibody or refund the original purchase. I look forward to your reply.

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