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Matching 2D membrane spots to the gel

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Matching 2D membrane spots to the gel

Postby samkh918 » Sun Sep 20, 2009 8:52 pm

Hello,

I am performing two-dimensional (2D) electrophoresis experiments and for each sample I make two IPG strips. After performing the first dimension for both strips, I stain one gel with coomassie blue to see all the spots, and use the other gel to transfer the proteins to a nitrocellulose membrane and probe the membrane with the antibody against my protein of interest.
When I see the single dot on the membrane, I don't know how to match it to the stained gel to show the location of my protein of interest among other spots.
I was wondering if there is any standard way of overlapping the antibody-detected spot with the coomassie stained gel.

Thanks for your help.
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Re: Matching 2D membrane spots to the gel

Postby JackBean » Mon Sep 21, 2009 8:39 pm

Hello,

I work on similar experiments. Our group works on 2D-electrophoresis of wheat and zea embryos. For this experiments I use methods of CUP-LOADING for 3-10 pH gradient. After IEF I work with second dimension in polyacrylamide gels. First gel with silver staining, second for Western blotting/for an one biological sample.

I make the Western blotting with nitrocellulose membrane and first POLYclonal antibody. For second antibody we use SWAR sera. After this proccess I stain the nitrocellulose with PONCEAU and then I choose some place of membrane. In my case 30-75 kDa. I display spots of marker by pencil and I continue in another steps. After the end of blotting I compare silver stain gel and film from nitrocelulose membrane. Used strip I give to film and I choose a place of presence my protein according to pI and Mr my protein.

Feel free to ask for more
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