I have just begun western blots looking at CD40 in immune cell pull downs. The antibody daasheet states the band will be apparent at 41kDa, although the predicted size is 34kDa. I am not too concerned about my blot, as I have just done a ponceau stain which showed a clear band around 35kDa (primary overnight to image tomorrow).
I just wondered whether anyone else had a similar experience with this antibody in particular?
Thanks very much!
dear Brittany,
I haven't experiance with the abcam antibody, but with CD40 and WB, and I can say to you that in my hands the better Ab for CD40 detection in WB is by Santa Cruz the H120 clone (I have used it on human cells). The right and only band I can observe is at about 44KDa and the observed MW higher than the predicted one depends on protein glycosylation. Moreover, to solve the problem of your WB in particular, I could suggest you to use different MWs from different brands because sometimes they behave differently and can lead to different conclusions.
Best and good luck
Gaetana
Hi Gaetana, thanks very much for your reply. I have had a second repeat over the weekend which showed similar results, so I may purchase the Santa Cruz antibody for my next run. When i've ran westerns on CD40 before with different treatments, the band was also at around 42kDa so to 35 just seemed too low. Thanks again for your help.
Best wishes,
Brittany
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