Yes it runs as a dimer in SEC. I run the protein by adding lammli buffer & boiling for 5mins.

I didn't perform mass spec.

Is it a soluble protein or a membrane one? I didn't get the fact that it is a homodimer and you produce only one of the domains, that would still mean that in solution your protein is a dimer no? Try heating for a bit longer (10-15 min at 70°C, or adding a bit more SDS into your Laemmli buffer).

Its a membrane protein which forms a dimer. The work with the whole protein is done & now I want to investigate the domains.

So, I have purified one of it's domains.

The protein itself forming a dimer doesn't mean that it's isolated domain will also form a dimer.

I will try your suggestion of adding a bit more SDS and heating a bit longer.

Thank you @Jorgaq Pata

What is the estimated isoelectric point of the protein domain you have cloned, and does it contain any negatively charged post-translational modifications?

The reason I ask is that not every protein (or domain) interacts equally well with SDS. Poor binding occurs in regions of high local negative charge (cluster of acidic amino acids, or of phosphorylation sites), due to charge repulsion. The end result is in an upward shift on the gel, sometimes substantially. For example, on SDS-PAGE, it has long been known that caldesmon (93.2 kDa) migrates at ~140 kDa.

So, if you have a similar negative charge cluster in your cloned domain, it may not make a difference how long you boil, or how much SDS you add, it might still migrate anomalously.

Dear Dr. Kent Arrell, the estimated pI of my protein domain is around 5. It does not have any negatively charged post-translational modification, however, it does contain more acidic amino acid residues as compared to basic ones. Thank you for your insights.

Dear Dr. Sebastian Schmitt, the molecular weight of the domain as estimated from SEC is of a dimer.

Yes, the protein is there in cell lysate only after induction and it produces quite a thick band as compared to the other bands in the lysate.

I would try out your suggestion of heating it to 70℃. Thank you so much.

So, in answer to your original question, yes, it can migrrate by SDS-PAGE at an estimated mass equivalent to its dimeric form, but this may be the result of other inherent chemical properties, not necessarily because it has formed a dimer. With a pI of ~5, this could certainly be the result of an inability of SDS to fully coat the protein at the usual mass/mass ratio (1.4 g SDS per g of protein).

Thank you Dr. Kent Arrell.

I'm guessing your expression construct is inadvertently tandem, so it codes for a dimer. Did you double-check the gene?

Hi Dr. Carter,

I thought of that in the beginning, so I cross-checked my construct sequencing. The construct is correct with my gene of interest flanked by the restriction cut sites.

Is it possible that the dimer formation of the protein is because of the domain? You can try running a native PAGE to check this, if you obtain a band at the size of a dimer, it may be possible that your domain forms dimers and this formation is not susceptible to SDS treatment.

Thanks, Aakash, I would try that. As of your question, the domain contributes to the majority of the interface of the protein dimer.

Tushar Chakraborty

Solubilized membrane proteins sometimes do not migrate to their proper mass, due to the presence of detergent around them, SDS is not able to substitute all the detergent molecules, and this gives an apparent molecular mass higher than the real one, however in your case, the difference is too much.

Try to put the lysate in a gel and/or western blot to conclude whether your protein is strictly dimeric in gel or no, even though your SEC gives an idea of that.

You may also try to denaturate your protein using urea/guanidine or other treatments to see if that changes something.

Dear Tushar, there are the possibilities that your dimer is not sensitive to the SDS. You may try with the different concentrations of SDS. In addition to the Native PAGE, you should also try with the zymography if possible.