SDS page gel GST beads or not to? - (Oct/08/2006 )

Hi all:

i've been doing some protein-protein GST pulldowns and wondering: after i boil up the glutathione sepharose beads in SDS and spin them down, i put the SDS solution into my SDS page gel. Should i also include the beads into the gel?

thanks.

Danny

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Hi Danny,

It doesnt really matter either way. Boiling in SDS buffer disrupts the non-covalent bonds between GST and the glutathione-sepharose-beads. Hence your GST-protein is located in the supernatant when you spinn down the beads.
On the other hand if you choose to load the whole mix (incl the beads) the beads will just hang in the gel slots and hence not really affect the electrophoresis.

Good luck!

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thankyou Dr. Dandelion

I appreciate your quick response. I'll try it out!!

thanks again!!

danny

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i don;t bother, just boil them together and load as it is. beads are much softer when boiled in SDS-sample buffer. i load all and never have problems.

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