Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

No WB bands for H2AX at all...! - (Aug/13/2012 )

Pages: Previous 1 2 

First, some antibodies are not strong for their antigens. So you have to use more primary antibody.

Second, what kind of ECL have you used? There are there kinds, week, regular and sensitive.
Base on our experiences, week antibodies do not show any bands with even regular ECL, like

Luminata Crescendo Western HRP Substrate from Millipore
http://www.millipore.../tech1/00112798
http://www.millipore...LE/00112798.pdf


We use regular ECL to have less overexposed bands. But sometimes, you have to use Sensitive ECL like this one from BioRAD:
Immun-Star™ WesternC™ Chemiluminescence Kit #170-5070
http://www.bio-rad.c...uminescence-Kit

Third, how much antigen are there on the PVDF? the less antigen, the more amount of primary antibody should be used.
I have even used up to 1:250 from primary antibody to see a band for an antigen.

Forth, also there are three kinds of Autoradiography Film, week, regular and sensitive.
We use this "HyBlot CL Cat#E3012 from http://www.denvillescientific.com/ " because it has less background.

-memari-
Pages: Previous 1 2