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Western blotting - (Nov/09/2013 )

Does the gel which is comassie stained can be used after destaining for western blotting????




I wouldn't use it.  If you want to check the efficiency of your gel, you can check your membrane post transfer using ponceau stain.


Typical coomassie stains fix the proteins in to the gel so they won't transfer.


Any time I've REALLY needed to coomassie stain before western, I just run two identical gels simultaneously...stain one and blot the other.


if you must use the stained gel then you can but...


assuming that you used a standard coomassie stain procedure where acetic acid is the fixative (not tca):


you must first wash out the destaining solution (methanol/acetic acid)


addition of 0.05% sds in the transfer buffer will help transfer (make sure you include 20% methanol, as well)


most of the coomassie stain will be removed during transfer


keep in mind that transfer efficiency (and maybe binding efficiency) will be different from that of an unstained gel