Dark western blot film - (Jul/04/2011 )
when I develop my PVDF membrane blot on Kodak X-ray film I get dark films. However, I do see my bands of interest. Only problem is that the film turns our very dark after developing and fixing.
how can i avoid getting a dark film??
sansub on Mon Jul 4 15:10:23 2011 said:
when I develop my PVDF membrane blot on Kodak X-ray film I get dark films. However, I do see my bands of interest. Only problem is that the film turns our very dark after developing and fixing.
how can i avoid getting a dark film??
I had this problem once, due to bad blocking in PBS with wrong pH (pH low)
Really? But, the pH of my PBS is correct.
blocking problem?
what is your blocking buffer?
what are your antibodies(antiphospho?)
Is this a new problem? Could it be that your film is just old? Seems obvious but I had that problem when I joined a new lab in which the film hadn't been used in a while and when I bought new film it made a huge difference.
it is insulin receptor antibody. blocking buffer used is skim milk powder in 0.1% Triton in PBS
was it an anti-phospho antibody? milk contains a plenty of phospho-caseins...
it is insulin receptor beta antibody from Santa cruz biotech. it is not an anti phospho Ab.
the secondary antibody used was HRP conjugated goat anti rabbit IgG
did you wash out the triton before to incubate with HRP substrate?