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Help to affinity purification with specific antibodies!!
I´m having some trouble purifying a certain protein...
This is my current setup:
I´m using pAb´s 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
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...
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?
|affinity , antibodies , purification , specific|
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