HSS Depletion Conditions for XKCM1¶
Contributed by Timothy Mitchison, Harvard Medical School, Boston, MA, United States
HSS Depletion Conditions for XKCM1 (Arshad Desai 3/17/95)
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.
- 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:
- Wash beads 2x with CSFXB +PIs.
- Wash beads 2x with TBST
- Wash beads 1x with TBS
- Add 50 ul SB w/ DTT.
- Also add 3 ul of each supe in 60 ul of SB
- Boil for 5’, pellet out the beads and transfer supe and freeze gel samples at -20 deg.C.
This method is based, with permission, on an original protocol available here.