You can use native PAGE along with molecular weight protein ladder for native PAGE. This will give you a rough estimate of your protein size.

Or as you mentioned, you can use a cross-linker and run the protein on SDS-PAGE. Check out this PDF: http://www.fgsc.net/neurosporaprotocols/How%20to%20cross-link%20proteins.pdf

It is possible to determine the MW of a protein by Sedimentation equilibrium analysis or by mass spectroscopy using differents techniques: Multiple-Charge Electrospray Mass Spectra;  matrix‐assisted laser desorption/ionization (MALDI)-MS; high-mass-accuracy time-of-flight mass spectrometry (LC/TOF-MS).

https://www.agilent.com/cs/library/applications/5989-7406EN.pdf

http://msf.ucsf.edu/documents/Strupat_MethodsEnzymology_v405

http://www.sepscience.com/Information/Archive/MS-Solutions/247-/MS-Solutions-16-Determination-of-Intact-Protein-Molecular-Mass--from-MultipleCharge-Electrospray-Mass-Spectra

Hi..

The rough estimation of the proteins can be examined by simple SDS-PAGE; Although, this technique gives discrepancies in MW as compared to other methods. Still the SDS derived molecular weights are valid. 

Hi Sneha thanks for your suggestion.. Actually I dont have native marker to run in the native PAGE. Thats why I was looking for alternate way.

Cross-linking and then SDS gel? Only a suggestion.

There are a lot of techniques existing, but I don't know what kind of material you have access to.

You can use DLS

You can use analytical ultracentrifugation

You can use high mass mass spectrometry

SDS page on cross-linked protein would give you some hints but would probably not be more precise than your GF experiments.

For your gel filtration, it should useful to have a protein marker with a MW close to 120kD to have a better estimation. The non  denaturing PAGE could give also some clue, but also in that kind of experiement a protein marker exhibiting more or less the same charge and MW should be sused to compare.

I would recommend SDS gels on crosslinked proteins - it is the simplest approach and will give much more accurate results than the GF as any potential charge effects on the proteins are negated as is interaction of the protein with the column packing.  

If you have access to a high mass MS system (Tof for example) that could also be of use but technically more challenging. 

  It seems that you already ran the calibration of the column. Moreover, you made the conclusion that the packing is perhaps not proper. Then, is it also not appropriate to use the calibration curve to measure the molecular weight?

  Based on the on-line document of the calibration of the Superdex 200 10/300 GL column which is for the analytic purpose, the calibration standards have M.W. of 6.5, 13.7, 29, 44, 75, 158, and 440 (in kDa, Gel Filtration Calibration Kits LMW and HMW). Moreover, the resolution around 158 kDa would not be great. I think under normal condition, your column should be fine to distinguish 39*3 and 39*4 kDa, however, because the “packing problem” was not clear at present, I cannot confidently say that anymore.

  If you would like to try SDS-PAGE with and without cross-linking or MALDI-MS, ultracentrifuge, those are fine. If you would like to try more about the gel-filtration of AKTA, you may want to include 1~ 5 mM freshly prepared DTT in the running buffer to see if there is any difference. Before doing so, I suggest trying the DTT concentration with a minimum amount of your sample before running the chromatography experiment. Personally, I prefer to see evidence from AKTA than from cross-linking.

Hi,

just a quick add to other answers. Gel filtration is not really a proper method for MW estimation, if your protein is not globular. For size (hydrodynamic radius) yes, but not for MW. The standards used for calibration are all globular proteins and as long as your protein is globular it is fine to use it for MW determination. However, if large part of your protein is unstructured the MW determined based on gel filtration can be few times higher that what it really is. Summarizing, if your dimer (39 kDa) is not globular it is not surprising that you see it as 120 kDa on gel filtration.

Regards,

Wojciech

DLS is a suitable method to determine the size of protein and then calculate the MW

DLS is a suitable method to determine the size of protein and then calculate the MW

I use a mix of several methods to determine the molecular weight of my protein. I used SDS first which confirms the molecular weight of the denatured protein. Then I used the following: 

Native and blue native gel  followed by second dimension SDS, and confirmed with western blotting.

DLS ( Dynamic light scattering), and if possible SEC-MALS, which mixes both gel filtration with multi angle light scattering.

I  also used AUC, analytical ultracentrifuge. 

All of those were combined to give an idea of the molecular weight of the native protein. 

as you are mentioning your protein is terameric/dimeric. your elution buffer plays a mojor role in its integrity. check your buffer Ph and ionic strength for protein to be intact in its form. And one you got your protein from the gel filtration you can concentrate your protein and run SDS gel. This might confirm whether its tetrameric or trimeric or single protein.

 @ Challagundle Naveen: Can I ask you please how can SDS confirm the tetrameric structure of the protein, and we know that it is rendering the protein to its denatured status? so we will definitely have it as a monomer on SDS. Or you mean as the others suggested cross linked protein then SDS! in this case I don't know this technique. 

Thanks to all for your kind suggestions. 

If you are going down the cross-linking route look at the Sigma and Fisher websites.

@sheera Abdulla, if its heteromer, we might expect different bands. unfortunatley if its is homo-tetramer then we might only single band.