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Affinity chromatography on NHS activated agarose with small peptides

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Tue Feb 19 04:52:01 EST 2013

Dear colleagues,
I have coupled a small peptide (1300Da) on NHS activated sepharose CL4B to affinity purify an antibody raised gainst this peptide. I get an unexpected low yield and when I re-apply the flowthrough on the column, again only a small proportion is bound. ELISA of the fractions supports that most of the antibody is in the flow through.

Does NHS activated sepharose have a size exclusion limit (the manual does not mention this) with the consequence that most of my peptide is trapped inside the resin where it can't be reached by the antibody?

Any suggestions to circumvent the issue?

Thanks for your help!


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