transfering proteins from 2-D gel to nitrocellulose membrane - (May/31/2008 )

Has any body ever tried to transfer proteins from 2-D gel to membrane in order to prob them with antibody? I want to do that but I'm not sure the SDS gel which we make for 2-D gel lets the proteins go into the membrane ! once I used this gel recipe for a regular western blot and it didn't work ...

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So far as I know you just treat it the same way as any ordinary western transfer. I am not sure why it didn't work in your case.

You can do partial electro-transfer of the 2d gel to the membrane (before staining), use it for western, and the gel with remaining protein you stain for getting 2-d spots. Next, you copy the western results on a transparency, adjust the size (in photocopier), to improve the co-localization of western spot with gel spot. (this is because the gel size will enlarge after staining and destaining).

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May I ask what is the material you use to make your SDS page and what is the recipe ... because I use the acrylamide/bis solution from Biorad and for large format 2-D gel we have to add another solution called strengthener to make the gel more durable, actually that was the cause of all the problem I had with transfering proteins from my gel the other time ...

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to blot 2D gels is a normal procedure; you may have to perform w/o the strengthener

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and what should be the transfer condition? I'm thinking about 200 mA for 4 hours for a large format gel to be transfered ... what do you think?

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best performance gives tank blotting O.N. but use PVDF as with NC you can blot through

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