Thanks for the all the reply my protein is filgrastim and is biosimilar to
Neupogen. it is stable at acidic pH of 4.0. i want to get rid of dimer
before i formulate it as we will be maintaining it in liquid form. What i
understand TCEP wont work. if i have to use SEC what buffer should i use. My
re
Does your SDS-PAGE loading buffer contain a reducing agent like beta
mercaptoethanol? That could be responsible for the difference between
your SDS-PAGE and HPLC results.
ho
On Fri, Mar 26, 2010 at 5:01 PM, CCP4BB automatic digest system
wrote:
> There are 5 messages totaling 490 lines in this
Yes, it is possible that the disulfide is just re-forming when the TCEP
is gone. pH 4 is not favorable for the oxidation, but does not prohibit
it either. Especially if there are traces of metal ions around. I
learned this the hard way, as trace metals can catalyze the oxidation of
methionin
Hi Ganesh and Mega!
I do not agree with Ganesh. I assume, Megha, that truly reversed
phaseHPLC was used. This is a denaturing method and the natural
disulfide should not form again during the run.
Also pH 4 can not be described "oxidizing". Actually, reduced proteins
are often dialyzed against
Hi Megha,
The two peaks on the HPLC indicate that your protein is
existing in a monomer-dimer equilibrium in solution. The dimerisation is
most probably caused by disulphide bridges. The use of TCEP is breaking
those disulphides and that is causing the equilibrium to move towards the
monomeric
Hi all,
My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce
it (directly added 1 M TCEP to make final volume of 10mM and kept at room
temperature for 10 mins] then i dialysed the protein sample to remove TCEP
[dialysis buffer is Na acetate pH 4.0].
On performing SDS PAGE a
