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 <[log in to unmask]> 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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