protein recovery from PAA gels - protocol needed

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kk
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protein recovery from PAA gels - protocol needed

Post by kk » Wed Mar 08, 2006 11:49 pm

I want to isolate my protein from PAA gel to sequence it. Does anyone have a protocol :?:


Some ideas I collected:
my protein has a C-term V5-tag, and it's secreted to the media. I will run the conditioned cell media through a V5-column, elute my protein, run it on a Western - this way supposedly only my protein will be in the gel. I stain the gel with a reversible stain (Ponceau), cut the bands out (i run several lanes with the same protein and pool them later), and extract my protein from the gel-piece. First I smash the gel, soak it in alkalic buffer pH10 for a day (not Tris buffer, because it interferes with the sequencing), spin, collect supernatant (protein fraction), dialize SDS and buffer out against PBS, liofilize my protein...for sequencing I need 50-100 pmol.


Thank u so much, peace!

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