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I'm having trouble using all abcam products with alpha smooth muscle actin antibody, ab 5694. I have attached a picture showing a ladder, skeletal muscle whole cell lysate (ab3869), and NIH3T3 whole cell lysate (ab7179). I loaded 10ug of protein into each well, 5% BSA block overnight, 2 hour of primary at 1ug/mL in 10mL TBS-T, 1:15000 secondary goat anti rabbit (ab97051), visualized with ECL+. 2 questions: 1) What muscle is the skeletal muscle lysate coming from, and should it have alpha-SMA? and 2) Why is my NIH3T3 lane not staining for alpha-SMA? Thanks!
Asked on Jul 06 2012
I apologize for the delay in getting back to you. I am still waiting for a reply from our source of the skeletal muscle lysate. I do not think it should contain alpha-SMA.
On the other hand, the fault may be with the antibody, if is not detecting this protein in the NIH3T3 lysate. I suspect that the antibody, though we have not received other complaints about it recently, is cross-reacting with something in the skeletal lysate that happens to be the same size as SMA.
Have you tried staining blots of these lysates with any other antibodies? If so, were the results as expected?
Answered on Jul 06 2012