12 replies to this topic

[sansub]

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??

[little mouse]

sansub, on 04 July 2011 - 07:10 AM, 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)

[sansub]

Really? But, the pH of my PBS is correct.

[little mouse]

blocking problem?
what is your blocking buffer?
what are your antibodies(antiphospho?)

Edited by little mouse, 04 July 2011 - 11:31 PM.

[knuf]

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.

[sansub]

it is insulin receptor antibody. blocking buffer used is skim milk powder in 0.1% Triton in PBS

[Inmost sun]

was it an anti-phospho antibody? milk contains a plenty of phospho-caseins...

[sansub]

it is insulin receptor beta antibody from Santa cruz biotech. it is not an anti phospho Ab.

[sansub]

the secondary antibody used was HRP conjugated goat anti rabbit IgG

[little mouse]

did you wash out the triton before to incubate with HRP substrate?

[sansub]

Yes, I washed it with PBST

[jopa]

I've sometimes get that with the Amersham ECL Advance Kit, I've found it to be very sensitive and gives a very high background, I see bands that are unspecific which I dont see with other kits, and sometimes I even seen the marker bands... overall I get very blank backgrounds...

I suggest you try another substrate kit for HRP and carefully dry the membrane of excess substrate...

[little mouse]

you should better wash twice with PBS without triton or tween before to put the substrate.