Dear Eleanor

I suggest you use SEC or SEC MALS  instead of PAGE for estimation of native configuration of your protein. Superdex 75 or Superose 12 may be a good option.

Hello Eleanor,

I would also suggest Size Exclusion Chromatography. You could also use Mass Spec to get an accurate mass for the oligomer.

Native gels are not intended for determination of protein size. They work best for finding out if your protein might bind to its ligand and change its migration pattern because of forming a complex. Alternatively, it is possible that a protein composed of heterodimers that run as single chains to bind a small ligand and migrate as dimers due to effects of the ligand. If your goal is to assess MW or mass of your protein, an SDS-PAGE is much better than native gels. Alternative more accurate methods are suggested by previous members with the caveat that SEC is rather crude in size determination as many other factors such as compactness, charge etc coem into play.  

Thank you all for your input. I have already done SEC on the protein, the results of which suggested it may be forming oligomers. So I am looking for another way to assess this, SEC-MALS sounds like a good place to go from here, thanks! 

An easy way to quickly check the "oligomeric state" (if the device is present) is dynamic light scattering .... SEC is also great in this regard, but for me it sometimes failed to tell me the "true oligomeric state" of some of my proteins (mainly because of dynamic eqilibria between e.g. monomer and dimer) ... However, the sure-fire method is MS.

If you want to determine the molecular mass of the species present in your solution, another method you could try would be to perform analytical ultracentrifugation sedimentation equilibrium experiments.  These are relatively quick (usually the data are collected overnight), and the analysis with modern software is easy enough.

SEC is best alternative, but can also try Blue Native PAGE.

You have to be very careful with membrane proteins on blue native gels, particularly if they are small. We addressed a whole bunch of issues in this paper: 

"Lipid, detergent, and Coomassie Blue G-250 affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers J Biol Chem. 2013 Jul 26;288(30):22163-73."

With this in mind a good starting point is Ref. 38: Wittig, et al.  (2006) Blue native PAGE. Nature Protocols. Note, gradient gels are more likely to give you the resolution you need. They can be a little tricky to cast yourself, though you can always purchase them as part of a BN-PAGE kit.

Eleanor, neither MS nor DLS are foolproof for oligomeric mass determination - DLS analysis relies on the proteins of interest, and their oligomers, being globular, while MS often disrupts oligomers and just gives you monomer e/M ratios. AUC is fine but quite cumbersome, laborious, long and difficult to analyze. I hope you have had a chance to test out SEC-MALS by now! It's easy and fast, and the folks at UManchester who run SEC-MALS are very knowledgeable and helpful.

An update -  the reason we suspected it may be forming oligomers is because it elutes much earlier off the gel filtration column than expected for a protein of its size. We did do SEC-MALS in the end which confirmed the monomeric state of the protein, and have recently also done some SAXS which showed the protein to have an extended dumbbell type shape, explaining its early elution during SEC..so native page was not the way to go after all (as many of you said!). Thank  you for your advice! 

I want to check the oligomeric state and interaction of small peptides with target proteins. Can anyone please suggest me which technique is best among Native PAGE, SEC-MALS or any other.

thanks