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-   -   Dark background on blots (http://www.molecularstation.com/forum/western-blot-forum/84311-dark-background-blots.html)

raghu.dvs 12-14-2011 06:38 AM

Dark background on blots
 
Hi,
I need suggestions, as my blots are too dark(high background) I can see my bands are weak hidden under dark background. MY blotting condition are
Transfer condition- 80 volts-2hrs (tris buffer 25mM, 192mM glycine 20% methanol)
Blocking buffer- TBST (0.1% tween20) with 5%milk powder- 2hrs
Primary antibody- 1:200 dilution in blocking buffer- overnight
Wash 3times 10min each in TBST
Secondary antibody- 1:3000 dilution in blocking buffer- 1hrs
wash 5 times in TBST and 1 time in TBS 10min each.
Exposure-ECL reagent (Luminol)..
The background is too dark..please suggest your comments.
Thanks

lyrachang 12-31-2011 11:25 PM

Re: Dark background on blots
 
Maybe you can use lower 1st or 2nd Ab concentration, or play around with incubation time?

lendaSoL 03-16-2012 01:29 PM

Re: Dark background on blots
 
hey.. try diluting the Ab more ( both the primary and secondary), or
increase the number of washes, or reduce the time of ECL on the membrane and try different exposure time..
good luck =)


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