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Dark western blot film


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#1 sansub

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Posted 04 July 2011 - 07:10 AM

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??

#2 little mouse

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Posted 04 July 2011 - 07:28 AM

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)

#3 sansub

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Posted 04 July 2011 - 09:39 AM

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

#4 little mouse

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Posted 04 July 2011 - 11:30 PM

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

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


#5 knuf

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Posted 05 July 2011 - 05:09 AM

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.

#6 sansub

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Posted 11 July 2011 - 08:53 AM

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

#7 Inmost sun

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Posted 11 July 2011 - 09:20 AM

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

#8 sansub

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Posted 11 July 2011 - 09:25 AM

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

#9 sansub

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Posted 11 July 2011 - 09:26 AM

the secondary antibody used was HRP conjugated goat anti rabbit IgG

#10 little mouse

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Posted 12 July 2011 - 02:07 AM

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

#11 sansub

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Posted 12 July 2011 - 05:14 AM

Yes, I washed it with PBST

#12 jopa

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Posted 29 July 2011 - 01:41 PM

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...

#13 little mouse

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Posted 17 August 2011 - 07:11 AM

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




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