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Thanks for help us to figure out this problem.
No signal in western blots:
HEK293, 50ug/lane, RIPA w/ PI, 100C, 5m. Markers transfer, 5% milk, 1/400 - 1/1000. Good 2ndry.
Asked on Sep 12 2012
Thank you for contacting us about this issue. Your protocol appears to be correct, and you should be seeing a signal with 50ug per lane and a 1/400 dilution. Sometimes switching from milk to BSA for blocking and diluting the primary will help develop the signal more but if you see nothing at all then I am skeptical this will help.
Have you been able to stain blots of these same samples with other antibodies? You mentioned that the secondary was known to be good but it is not clear if you used with other primaries on these HEK 293 lysates, or different lysates. In any case, I think a replacement is in order. Are you interested in trying a polyclonal antibody instead, such as ab82383?
I look forward to your reply.
Answered on Sep 12 2012