Huge background with HT22 cells phophoERK antibody - (Dec/12/2011 )

Hi all,

I am studying the ERK activation in HT22 cells and for this first i probe my blot with total ERK and got good signal and later on when i do stripping. After stripping the blot i prob the blot with phosphoERK antibody.

My total ERK gives very good signal, while there comes huge background in the phospho ERK probing.

Can anyone gives some suggestion.

thanks

Always do the Phospho's first and then do the total....Phosphorylations would get affect on stripping...background might be due to various factors including the antibody dilutant...sometimes if there is no detectable phosphorylation on blot would give backgrounds....hope your lysis buffer has the phosphatase inhibitors, have a check at this also...

phospho ERKs generally i keep for overnight at 4centigrade,then it works good.myself uses 1:1000 times dilution primary
and 50ug protein/well.and ofcourse its always good detect phospho ERKs first or else use 2 different gels or blotts for ERK ans phosph ERK.

with what do you block (and add to your antibody solution)?

if you use milk then that could be the cause of your background. milk contains phosphoproteins (e.g. casein).