i was doing it for PCNA and BAX...Santa Cruz antibodies...1:500;;;PVDF membrane and used both biorad transfer turbo and imager......but they worked with me before??
Hi, check your protein concentration - it could be too low, or did not transfer adequately.
Check your solutions dates of expiry.
Look for any small detail that might have changed since the last time it worked.
First, blot your membrane with beta-actin. If you get the band, then your transfer is fine. Try to use higher concentrations of antibodies or avoid using Santacruz antibodies. Abcam or cell signaling antibodies are good. If you are doing in tissue make your sample in RIPA buffer and then add sample buffer.
These are really very helpful suggestions!!
i will try them hopefully avoiding this situation
Thanks so much
stain your membrane after transfer with Amido black. If you have protein transfer then you will see it. You can then go on and probe for your protein...including a loading control like actin or tubulin.The amido black stain will tell you is its a problem prior to transfer or an antibody/blocking issue.
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