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-   -   Help to affinity purification with specific antibodies!! (http://www.molecularstation.com/forum/antibody-forum/86803-help-affinity-purification-specific-antibodies.html)

vivaspin6and20ml 11-13-2012 11:00 AM

Help to affinity purification with specific antibodies!!
 
Hi..

Im having some trouble purifying a certain protein...

This is my current setup:

Im using pAbs that are covalent coupled to protein a - sepharose beads (~ 1.5 mg ab to 1.5 ml 100 % beads) with glutaraldehyde.

Incubate with ab for 2 h at RT followed by fixating with glutaraldehyde for 1 h RT and neutralize with ethanolamine (~ 400 mol excess)

By calculating the remaining ab from coupling incubation, all but 0.08 mg was bound.

The beads are placed on a 20 ml column from Bio-rad and washed 5xCV in PBS pH 7.4

Incubate with serum (10 ml) in 10 ml buffer over night at 4 degrees on rotation

wash x 5CV in TBS + 0.05% tween 20

elute bound protein with glycine 0.1M pH 2.5 and neutralize with Tris-HCl 1M pH 8

RESULTS:

ELISA:
Shows that my protein elutes i certain fractions, and that there is a profound difference between in the amount of protein in the initial serum sample and serum after incubation on the beads...

WESTERN BLOTTING:
Shows that my protein elutes in the same fractions as indicated by my ELISA

COOMASIE STAIN + SILVER STAIN:
Shows rather weak bands around the theoretical molecular weight of the protein that aligns with what I see in the WB, and as indicated by my ELISA.

HOWEVER I get substantial amounts of other proteins in the elutes...!!!

How can I avoid this?? I figure that it must be either unspecific binding into the seph-beads, cross-reactivity of abs with my coupled pAbs, the pAbs I use recognize other serum proteins aside from my desired protein or that the protein a on seph-beads are not saturated, and therefore binds serum abs, which recognize other serum proteins...

Is it possible to wash these "other" proteins away and get a more pure product?

Please help..

Thanks


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