I am using MAbTrap Kit with HiTrap Protein G HP 1 ml column(Amersham),
to purify a recombinant human antibody (IgG1). This antibody is
expressed in adherent CHO cells. I am using DMEM with 5% SYNSER. I am
not using any FCS in culture. SYNSER is a Hybridoma growth supplement
from Biological Industries, Israel. I am facing the following problem
I performed an ELISA to check antigen binding with different fraction.
I used equal amount of each fraction for the ELISA ( 0.5 micro gm
/well). I measured the protein content of each fraction both by OD280
and by Bradford reagent BioRad.
As far as I understand the purified eluted fraction should give more
ELISA reading than the original crude sample.
It seems, I am loosing lots of the antibody. But OD280 of Wash
fractions were near zero.
How can I increase the yield of purification?
Can the neutralization buffer, used during purification, creat problem
Expecting your suggestions.
Biplab [Only registered users see links. ]
Department of Biochemistry
All India Institute of Medical Sciences
New Delhi, INDIA
Ph.No: 2659-3314 [Only registered users see links. ]
Could be that something inhibits binding to the column. IgG1 should bind
perfectly to protein G actually (just checked it), for a nice chart see
[Only registered users see links. ]
You might try ammonium sulfate precipitation of your cell supernatant in
order to remove small interfering molecules prior to any chromatography.
If you like, give us more details about the construct you're expressing and
your ELISA protocol. Could it be that there is something wrong with the
sequence (incomplete, frameshifts, mutants, no secretion signal sequence...)
You might read this papers by Roffler-S on purification of engineered