Can I simply add ECL again? - (Oct/04/2006 )

Hey all,

I have a blot, everything went smoothly with clear bands etc.--however, I think, upon looking at it, that the ECL may have been added unevenly (there are patches of a few bands in a row that are oddly light, and I don't think it has to do with amount of protein loaded, loading control looks good...)

My question is, can I simply wait awhile and just add ECL to the membrane again and re-expose, or would I need to strip the membrane and re-add primary/secondary/etc.?

We're trying to do a relative quantitation, and obviously can't do that if lightness/darkness of the bands has to do with ECL and not protein expression

Thanks for any help!

Hi tezzums,

My understanding (may be wrong) is that in the ECL reaction the horse raddish peroxidase is used up in the reaction, consequently if it was due to uneven coverage of ECL reagent than you would probably find that the light areas will become dark and the dark areas light. I'd strip and reprobe. Also I tend to find that more often than not these uneven banding is really an indication of transfer effeciency and therefore the samples may have to be run again. But I'd try stripping and reprobing first.

Cheers,

Scott

if you haven't allowed your blot to dry then you could try adding fresh substrate without stripping and reprobing. the hrp should be fine if it hasn't been dried (and maybe even if it has).

worth a try.