Sunday, October 29, 2006

Queen of Acrylamide Gels

One of my favorite protocols in the lab is running SDS-Page gels. Boss thinks Im nuts (I guess other people hate running them), but I kick ass at them so he lets me be giddy.

The all knowing Wiki proclaims that SDS-Page is: a technique used in biochemistry, genetics and molecular biology to separate proteins according to their size (length of polypeptide chain or molecular weight).

We're trying to separate out three proteins HIV makes-- gp160, gp120, and gp41. Gp160 is just a precursor protein, it gets chopped into the two bits that HIV really wants: a transmembrane protein gp41, and gp120, which noncovalently bonds to gp41 to create the spikes that stick out of HIV that attach to your cells.

(from 'Molecules of HIV')

The thing is, when we blow up the cells in our Pulse Chase experiment, we collect all the proteins the cells make, not just the HIV proteins. So we do something cool called an immunoprecipitation-- we add HIV antibodies to the cell goo, the Ab stick to the HIV proteins, and we add beads to the goo that stick to the Ab. When we wash everything off, only the beads, the Ab, and the HIV proteins are left! Then we boil the samples to denature the proteins (they need to be linear to run in a gel) and BAM! Pretty gel time!


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