Protein G Sepharose beads preparation - (Aug/25/2009 )

This may be a bit basic but could any one help me to understand the procedure for making a 50% slurry with sepharose beads? They came packaged in 20% EtOH and believe I need to prep about 400mcl of slurry for use in a Co-IP experiement.

I believe I just wash the beads in my lysis/IP buffer and reconstitute them in a certain volume but not quite sure all the logistics.

Thanks

dna_nerd on Aug 25 2009, 08:02 PM said:

Yes that's right, I usually wash 3 times to make sure I get rid of all the EtOH, then to make a 50% slurry just resuspend the beads in an equal volume of buffer i.e. to 200 ul beads add 200 ul buffer.

P

Penguin on Aug 26 2009, 08:38 AM said:

I transfer a portion of a bead-EtOH slurry to a new tube, centrifuge, aspirate the supernatant liquid, and wash 3 times with 20x the volume of cell lysis/IP buffer, then pre-block with 10% milk in cell lysis/IP buffer. Finally, pellet and resuspend the beads in an equal volume of buffer.