Depends on how it's dimerised. Cleavage might be an option.

You can always boil it too (10min at 98C will probs do the trick).

Can you give some more details of what you need to achieve?

Dear Alexander & Miral,

thank you for your valuable suggestions.

I did the same thing, i added BME and then boil it at 94 degree centigrade for 10 minutes but i Could not find the single band.

I am trying to purify Chikungunya Envelope protein that is coming in inclusion bodies, and after following inclusion bodies purification protocol, I performed affinity chromatography (Ni-NTA, His-Tag)by FPLC to purify the protein, then I run it on SDS-PAGE, and I found the dimer.

Then I tried to remove dimer by adding BME to remove dimer but it didn't help.

Dear Sebastian,

Thanks once again for your answer

you have given some valuable suggestions for my previous question and I followed the same protocol suggested by you and found the same band as I got earlier and yes, you are right, I am not so sure that this is my protein of interest, so I will do western blotting and Mass spectrometry to confirm whether this is the protein what I desired or not.

But for the information I want to know any other method to remove dimer without using BME.

Dear Sebastian,

Thank you for the suggestion, I will try to do the same at lower temperature.

Amended on 13 June 2018

I have experienced that hydrophobic-glycoprotein enzyme human-serum biotinidase (serBIN) is present in serum as dimer and monomer forms in the presence of 2-mercaptoethanol (2-ME) (please see file; BIN Mr 110000).

Finnish Biochemist Dr. Jaakko Pispa has indicated that serBIN is Mr 115,000 as determined by SEC without using 2-ME and Brij-58 in the report of

Pispa J. Animal biotinidase. Ann Med Exp Biol Fenn 1965; 43 (suppl. 5): 5-39.

Therefore, 2-ME is not sufficient to get monomer, and further addition of non-ionic detergent of Brij-58 (at 1%(v/v)) may be necessary.

Further, although HPLC-Surf-SEC is suitable to separate hydrophobic proteins (please see file; SEC column 300 A silica), protection of thiol groups by pyridyl-ethylation (PE) may be recommended.

Thiol-protected monomer may be also safely separated by our RP-HPLC method (please see file; Lysozyme by RP-HPLC).

Analysis of hydrophobic proteins by SDS-PAGE is not recommended at all (please see file; IEF for hydrophobic protein).

Have you tried TCEP as the reducing agent instead of BME and DTT?

It is worth noting that most commercial precast gel vendors actually do not recommend boiling (or close to 100°C) anymore, based on the fact that boiling actually promote dimerisation/multimerisation on some membrane proteins.

Deepan's suggestion is solid. TCEP is a far stronger reducer than bME and DTT, with higher stability. Also perhaps adding a bit of reducing agent in the PAGE running buffer? as per Sigma TruPAGE system manual. All the recipes are described so no need to purchase anything.

Lastly, the real question is, as asked already, are you sure this is your protein? If that is certain you could always mutate the predicted dimerisation residues to see the band shifting. And it generates a pretty figure for publication too.

Dear Deepan & Ian Hu,

I did not try TCEP because it is not present in our lab, but if it can be useful, I will try this also.

Thanking you

Ankur

I agree with Alexander. He has rightly answered your query.