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Antibody Affinity Purification


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#1 Danio ivanio

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Posted 30 June 2011 - 04:22 AM

Hey there,

we ordered a antibody and recieved the anti-serum. Now I did the affinity purification with the peptide column. After washing the column with PBS pH 7,4 I eluted the antibody with pH 2,7 Glycine in Eppendorf cup containing 50µl of Tris pH 9. When I measured the fractions with Nanodrop, I prior blanked with Glycine+Tris...The values are really messed up and jumping high-low without any sense. Does anyone has an idea why? Should I use convenient Spectrophotometer with Lowry or Bradfort rather than Nanodrop? Any suggestions are appreciated.

Thanks
Ivan


Edit: I ask myself...Should I do size exclusion column directly after affinty purification, somehow I see a small pellet in some fractions...perhaps tris and glycine are precipitating my antibody. Desalting and changing the buffer to PBS should do the trick maybe...then try nanodrop

Edited by Danio ivanio, 30 June 2011 - 07:34 AM.


#2 BioMiha

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Posted 30 June 2011 - 09:13 AM

If there were a lot of antibodies in your antiserum, then precipitation is quite likely. That would also explain why the OD measurements were all over the place. I would definitely dialize the eluted fractions, however I wouldn't apply the pellet to a SEC column as it might stop the flow. Use a dialysis membrane instead. Some Abs have a pI close to 7 so they precipitate in PBS as well. In that case a buffer with a lower pH is better.

#3 Danio ivanio

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Posted 30 June 2011 - 10:21 PM

Thank you for your advise. :)

Ivan




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