Procedure:

(This protocol uses anti-XKCM1 for immunodepletion.)

Put 25 ul of Bio-Rad Affi Prep bead slurry into two 0.5 ml tubes labeled IgG and XKCM1. Wash beads 3X with 0.5 ml TBST each wash. Add Rb IgG (4 ug) or anti-XKCM1 Gly (4 ug) and bring volume to 100 ul total. Bind antibody to beads at 4 deg.C for 1 hr 15' on rotator. Make sure beads are rolling around. Pellet in ufuge in coldroom and wash 1X TBST, 3X CSFXB + PIs. Add 150 ul of clarified extract to each tube. Rotate for 1 hr at 4 deg.C ensuring that beads are mixing well. Pellet and transfer supe to a different tube. Aliquot and freeze 20 ul aliquots in green tubes (XKCM1 deplete) and in yellow tubes (IgG deplete). Processing beads for gel a) Wash beads 2x with CSFXB +PIs. b) Wash beads 2x with TBST c) Wash beads 1x with TBS d) Add 50 ul SB w/ DTT. e) Also add 3 ul of each supe in 60 ul of SB f) Boil for 5', pellet out the beads and transfer supe and freeze gel samples at -20 deg.C.

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Note: HSS is less sensitive to activation - I find it to be very stably CSF. Other types of protein A beads can also be used for this purpose - it is not necessary to use Affiprep beads. I have succesfully scaled up this depletion to 500 ul of extract increasing amount of beads and antibody proportionally.

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