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A lot of background noise on anti-phosphotyrosine blot?

western blot phospho-tyrosine py20 noise problem

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#1 DrZeus

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Posted 27 February 2013 - 04:24 AM

I have done several western blots with PY20 (mouse monoclonal) as the primary antibody with anti-mouse as the secondary. I block in 5% BSA in TBS overnight then incubate in the primary antibody at 1:20,000 dilution for 2 hours, wash in TBST and then incubate for 2 hours in secondary antibody at 1:2000 dilution. I keep getting a lot of background noise and have tried to play around with the dilutions/sample size loaded but still hasn't got better. Has anyone had any similar issues and if so how did they resolve them? Also, are there any things to look out for when using the PY20 antibody?

Thank you :D

#2 GNANA

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Posted 27 February 2013 - 05:34 AM

i am used to pY from millipore, i always block for 1 hr using 5% BSA/TBS, then one hr primary (1:1000) at RT, washes and 1 hr secondary, it work good....

i sometimes get black background wen i IP'd less protein and so got to expose more time to get detectable signal. so suggestion is less the time you expose to the film and get signal ll avoid you frm getting background. so the more tyrosine phosphorylated protein on the blot 'll reduce the exposure time and so ll reduce the background as well.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....





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