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 acidic buffers to prevent disulfide formation
via the thiolate anion.
Still, a reducing agent may be used during the run?
Sorry that I can not offer a solution to the real problem. More
experimental details may be necessary.
Bets regards, Matthias
Am 3/26/2010 8:37 AM, schrieb Ganesh Natrajan:
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 state. However, when the TCEP is dialysed out, the
disulphides start forming again and this is causing the equilibrum to
move towards the dimeric state, a process clearly hastened by the
strongly oxidising pH 4 of the dialysis buffer.
Now it all depends on what you want to do. If you want to use the
protein in a (largely) monomeric form, I would recommend that you
don't dialyse out the TCEP.
regards
Ganesh
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Blow, blow, thou winter wind
Thou art not so unkind
As man's ingratitude;
Thy tooth is not so keen,
Because thou art not seen,
Although thy breath be rude.
-William Shakespeare
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On Thu, 25 Mar 2010 22:51:30 -0800, megha goyal <[email protected]>
wrote:
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 analysis after dialysis of the protein we
are getting no dimer band, only band of our protein is observed
and same is the case with UV reading there is no change in it. But
the HPLC analysis of the protein shows two peaks instead of one
peak as observed before TCEP treatment. what can be the reason for
this. Kindly guide. i need the protein to formulate and conduct
stability studies on the sample. the protein we obtain after IEX
is pure except the dimer and i do not want to go for SEC as it
greatly reduces protein content and also is quite time consuming.
any light on what is happening will bevery useful.
thanks and regards
--
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****************************************************
Dr. Matthias Zebisch
Universität Leipzig
Biotechnologisch-Biomedizinisches Zentrum
Strukturanalytik von Biopolymeren
Deutscher Platz 5
04103 Leipzig
Germany
Phone: 0049-341-97-31323 (lab) -31312 (office)
Fax : 0049-341-97-31319
email: [email protected]
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