2 replies to this topic

[ANCO]

Hi all,
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.

[Crymych]

Try using FLAG peptide from Sigma. Product number F3290. I'm about to use this for my elutions at 1 mg/ml.

[mcgpostdoc]

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