2 replies to this topic

[dna_nerd]

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

[Penguin]

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

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

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

[Dr Teeth]

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

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

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

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

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.