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Buffer exchange of RecA protein


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#1 Luke William Cuculis

Luke William Cuculis

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Posted 09 July 2012 - 04:52 PM

Hi all,

I am trying to label commercially available RecA protein from EpiBio. From Epibio, the protein comes at 5 mg/mL in a storage buffer with 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100. I need to get it into a labeling buffer that is free of amines (doing amine labeling), so I am trying to buffer exchange it into 50 mM KH2PO4/K2HPO4 (pH=7), 1M NaCl, 0.1 mM DTT and 10% glycerol. I have tried using Millipore Amicon filters and Bio-Rad P-6 microspin columns (both mini centrifuge spin methods) for this buffer exchange, however it seems every time I try to do a buffer exchange the protein loses biological activity (ATPase assay is my measurement). Any thoughts on why?




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