Isolation of IP complexes - (Nov/27/2003 )
I'm now using M2 Anti-Flag affinity gel(beads) from Sigma to pull down my IP complexes but don't know how to isolate those complexes from the beads without denaturing the proteins .(This beads bear a Flag antibody that recognize the fusion protein at N-terminal, Met-N-terminal and C-terminal).Normally,we use elution buffer like SDS sample buffer to elute the complexes before applying to a Western Blot.As far as I know SDS may denature the proteins and I really need the natural form to proceed to another assay,besides the usual WB.
I'd appreciate if anyone could give me an advice.Thanks.
Try using FLAG peptide from Sigma. Product number F3290. I'm about to use this for my elutions at 1 mg/ml.
you can try the classical method of using a gradient of glycine hcl ph 2.5 or buffers available from pierce, elute your proteins in 1M tris