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Isolation of IP complexes


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#1 ANCO

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Posted 27 November 2003 - 07:57 AM

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. :)

#2 Crymych

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Posted 15 December 2003 - 09:46 AM

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

#3 mcgpostdoc

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Posted 23 February 2004 - 08:31 AM

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




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