Protein G purified IgG>>> extra band at approx 200 kd - (Jan/03/2008 )
Can anyone please tell me what is the extra band( 200 kd approx) other than the heavy chain and light chain at 50kd and 25 kd respectively coming when i purified IgG from culture supernatant. i ran 12 % SDS under reducing condition
can u tell me what is the % of intenisty of 200kDa band compare to ur antibody bands?
i wud suggest u to pay attention towards the source of ur protein G...
is it recombinant pro G beads or native one.
if it is native one then there is a possibility that it will bind to to serum albumins (as u grow hyridoma in serum containing medium) which can partly explain the extra band.
if ur protein G is recombinant one which does not have albumin binding site then, u can think about other possibilites, like some thing wrong with ur beads (too old) or multimerization or inefficieny of some buffers.....
[quote name='donot lie for ever' date='Jan 3 2008, 06:45 PM' post='121639']
thanks for ur reply.
the xtra bands is very thin and sharp..intensity is more compared to the antibody band.
If it is of serum albumins why it is coming in that range?? it should be around 60- 70kd
and i did not understand about the multimerization..
thanks for ur suggestion
i will also try to think in ur direction,
hoping to get an answer for this :-)
thanks once again
find out about any albumin protein which is around 200kDa of mw, which cud possibly bind to ur protein G....
even i used to get an extra band at the range mentioned by you when only purification is from culture supernatants. When purification is from other sources that band is not to be seen. I couldn't find out what is that band though.
elute the 200kDa band and send for mass spec analysis, if you really want to which protein is that.....