Hi Again my friends,
Just to recap; I had a problem with eluting off my antibody of interest from protein G column. It seems as though the antibody is bound tightly to the resin and is very difficult to elute it off. I've tried 10mM Na-phos+50mM NaCl @ pH 3, 2.5, and still the antibody peak absorbance on AKTA-purifier is 1/10 of that of what was initially loaded (starting material)...
the gel analysis for flow-through and eluate show no band at ~148kd (where antibody often shows up) and a clean band at ~148kd. But the quantitation is again, 1/10 of the original starting material.
Furthermore, the post washing of the column with 1% phosphoric acid shows a large peak on the chromatogram which most probably is the dead antibody coming off...
My alternative method is either to use ion-exchange chromatography instead of affinity.
However, before that, i want to try using 0.5M arginine buffer to elute off the antibody. i know there is some literature which suggests arginine elution with prot. A column works well; but what about prot.G??
If anyone has any experience using arginine for this purpose and can give me some feedback i would appreciate it.
Thanks in advance.