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Western blotting


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4 replies to this topic

#1 samita

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Posted 09 November 2013 - 02:36 PM

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

 

regards



#2 jerryshelly1

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Posted 09 November 2013 - 05:39 PM

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.



#3 bob1

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Posted 09 November 2013 - 11:44 PM

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



#4 labtastic

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Posted 11 November 2013 - 05:33 PM

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


Edited by labtastic, 11 November 2013 - 05:34 PM.


#5 mdfenko

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Posted 12 November 2013 - 04:36 AM

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


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