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Problems with affinity purification of anti-peptide rabbit polyclonal antibodies using Pierce Resins
Edith Blackburn
Monday, 02 April 2007 14:14 UTC
Our lab has spent many years purifying rabbit polyclonal anti-peptide antibodies from serum with good yield using Reacti-Gel HW-65F from Pierce.
These beads have a particle size of 32-63um and were used in affinity columns that were made by reacting the beads with the peptide of interest passing the anti-sera through the column then eluting the bound antibody with 100mM glycine buffer pH2.2
The problem is we can no longer buy this product. It is no longer supplied by Pierce and we cannot find an alternative supplier.
The replacements sent by Pierce have been Reacti-Gel(6x) Support and Reacti-Gel (GF-2000) neither of which have given very satisfactory results.
The yield is very low and with some antibodies no purification is possible at all. I have also tried the Pierce AminoLink Kit with ready made columns and reagents but none compare to our original Reacti-Gel.
If any-one has had similar problems or can offer any suggestions or advice or information as to where we may locate our original beads or something similar I would love to hear from you.
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Replies
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try uing CNBr activated sepharose 4B fast flow by coupling the peptide and purifing the Antibodies. If you have any better suggestion please let me know. as i am also doing the same thing but purifing the anti-peptide andtibodie fropm Rabbit is difficult by peptide affinity column.
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I have had better success recently using the new AminoLink Plus Kit from Pierce.
Again results not as good as the original beads but better than the other matrix offered by Pierce as alternatives.
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