Dimer elimination from protein

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exec
exec's picture
Dimer elimination from protein

 
My protein is 19 KDA protein and has a dimer of 38 KDA. My protein is relatively purified except the dimer part that can be seen in non-reducing page. i want to get rid of this dimer. can it be done by dialysis as Gel permeation does not work for me due to such small difference in their molecular weight.
Please provide me guidance on how shall i go about it.
Thanks in aniticipation
 
Exec

kriodos
kriodos's picture
hi

hi
A) first at all. Are you sure is a dimer of your protein? did you do a western for see if is a dimer of it or is a dimer of another?
B) Usualy the proteins taht dimerize do it in a condition. Them if you purify your monomer but matain the conditions your protein is going to dimerize again.
C) Do you know wich kind of unión do you have between the dimer? If the dimerization is by charge you can add NaCL, if is by disulfure bridge you can add DTT or b-Mercaptoetanol and have only the monmer.
D) Anyway you can sepatare proteins of diferent molecular weigt by molecular exclusion chromatography
for what do you need the protein?
 
exec wrote:

 
My protein is 19 KDA protein and has a dimer of 38 KDA. My protein is relatively purified except the dimer part that can be seen in non-reducing page. i want to get rid of this dimer. can it be done by dialysis as Gel permeation does not work for me due to such small difference in their molecular weight.
Please provide me guidance on how shall i go about it.
Thanks in aniticipation
 
Exec
exec
exec's picture
i need the protein for

i need the protein for filling into vials and carry stability testing of samples.
my protein is a dimer as we have checked by western blotting. size exclusion does not help much due to very small difference in the Molecular weight i.e 19 KDa and 38 KDa.
i suppose the dimerisation is by S-S bond as in SDS pAGE in non reducing we see dimers but in reducing condition only pure protein is observed. DTT addition as suggested what conc will be suitable as the ph of our protein solution is 4.0

qiang wang
qiang wang's picture
50mM DTT would help. But not

50mM DTT would help. But not suggested
I will suggest 1-5mM (I used before 1mM) b-mercaptoethanol, which works better, and moreover, DTT will mass up many assays.
If you want it non-reducing, and do not mind the denatureing condition, add 6-8M urea will do.
Or, if it takes a while for your protein to dimerize, still add urea, use the protein immediatly after dialysis the urea out.
 
hope it helps, and please do let us know how it works

kriodos
kriodos's picture
qinglongyanyuedao wrote:

qinglongyanyuedao wrote:

50mM DTT would help. But not suggested
I will suggest 1-5mM (I used before 1mM) b-mercaptoethanol, which works better, and moreover, DTT will mass up many assays.
If you want it non-reducing, and do not mind the denatureing condition, add 6-8M urea will do.
Or, if it takes a while for your protein to dimerize, still add urea, use the protein immediatly after dialysis the urea out.
 
hope it helps, and please do let us know how it works

Yes, 1-5mM can be ok. Have the protein at ph=4.0 is very good for avoid the formation of new disulfure bonds after reduction. Below pH 8.0 the SH groups of cystein are usually protonated avoiding the formation of disulfide bond. I hope this works if not tell us
good luck

kjosephs
kjosephs's picture
To reduce the disulfide bond,

To reduce the disulfide bond, 5mM DTT or BME is a good place to start.  You should be aware that these reducing agents will oxidize over time so the dimer could be reforming when stored for long time periods.  TCEP is more stable, but in some cases may still require that it be added fresh after long term storage.   If the protein is active and you need this activity preserved, you should avoid using urea.  Once you have reduced the dimer, lowering the pH can prevent it from reforming, as Kriodos suggested.  Though again, not all proteins remain active at pH4 so you may have to settle for pH6.0.  Beside lowering the pH after disulfide reduction you can also prevent dimerisation by keeping the protein at low concentration and including higher salt concentrations in the buffer, like 0.5 M NaCl.