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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Dark background on blots

Dark background on blots - Western Blot Forum

Dark background on blots - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 12-14-2011, 06:38 AM
Pipette Filler
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Default 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
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Old 12-31-2011, 11:25 PM
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Default Re: Dark background on blots

Maybe you can use lower 1st or 2nd Ab concentration, or play around with incubation time?
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Old 03-16-2012, 01:29 PM
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Default 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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