I suggest that if you want to separe monomers from dimers/multimers, the best technique that you can use is gel filtration (using HPLC, for example). With ion exchange chromatography you may obtain a population of dimers/multimers dependind on the buffer conditions (reductor agents, ionic strenght, and so on) and the dynamic of formation of those dimers/multimers, but, it is not the best technique to obtain those dimers/multimers

One would expect that with the surface masked by homodimerization the dimer will have a different total charge surface than the monomer, but that will not tell you if the minimal necessary surface for resin binding will be affected.

Therefore IEX may or may not be resolutive but you certainly cannot predict it from your protein sequence alone.

I mostly agree. Dimerization should not radically change the total charge of the molecule, but it is not completely impossible to have charge difference enough for separation. Although I do not have experience on my own, I would propose HIC as potential method of choice for separation of oligomeric species.

What is the size if the protein?

Hi,

actually you can separate monomers from dimers/oligomers of proteins by using ion-exchange. This will be (as usual) protein dependent but things are more complicated on the molecular scale than one would think, therefore I would not expect that because the (theoretically) overall charge distribution of a dimer could be similar to a monomer, that it would not be possible to separate them. As an example, we have separated monomers and disulfide-bridged dimers of PDGF by ion-exchange (confimed by non-reducing SDS-PAGE).

Ion-exchange is often underestimated and quite powerful in many cases and definitely worth testing. In simple cases when the size difference is significant, a gel filtration run could however be more straightforward.

In general, gel filtration is recommended.  In some cases, MonoQ column (GE Heathcare) can separate the proteins with the same net charge but different conformation.

It is important to know and consider whether you are dealing with covalently linked species (e.g. by disulphide linking) or with a purely noncovalent equilibrium. If it is the latter, then the rate at which the equilibrium adjusts is important. I agree with Francisco Rivera  that gel filtration is the most obvious method to use, but if you are dealing with a noncovalent equilibrium then speed may become critical and FPLC is indicated. Even so, with a rapidly adjusting equilibrium you may see skewed and/or overlapping peaks. At the extreme, if the components are reequilibrating much faster than you are separating them, a mixture of dimer and tetramer could appear to run as trimer!

Thanks for all your answers, really appreciate this! :) I have an FPLC system at my disposal meanwhile. I am trying to purify an aggregation prone molecule (self-interacting, no cocalent linkage between monomers)- since my functional assay includes measuring aggregation kinetics, I need pure monomer and therefore want to separate the dimers from the monomers.

Maybe in that case it would be worth, instead of  investing effort in separation, to try to explore buffer and salt conditions that would suppress the aggregation. How to do this will depend on whether you think aggregation in this case is primarily a hydrophobic process or an electrostatic one. If you don't suppress the aggregation, how long would it be before your separated monomers aggregated once again?

Hi Peter, are you working with an amyloidogenic peptide or protein?

I meanwhile established a buffering system that keeps/should keep my protein in solution in a monomeric form.

Do I recall correctly that ionic strength should be low at the start/binding of IEC?

And another question: How does flow speed effect the separation? Should I run the gradient fast or slow?

@ Steingrimur Stefansson: Yep, you guessed correctly - Are you also working with this fun stuff? ;)

Hi Peter, I was working with this stuff and enjoyed every minute of it/sarc.

Anyway, the peptides I was working with were ab (1-42 and 1-40), amylin and hamster prion. 

The most common way to get many amyloid peptide multimers into monomers is to treat them with organic solvents, such as DMSO.

http://www.researchgate.net/publication/232614087_Specific_Binding_of_Alzheimers_Abeta_Peptide_Fibrils_to_Single-Walled_Carbon_Nanotubes/file/d912f5087fdc6e5fd3.pdf

(and references therein).

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0056467

http://www.pnas.org/content/104/8/2649.long

Personally, I think that trying to isolate amyloid monomers in aqueous solutions is going to be problematic because it is hard to verify that you have monomers to start with.

I recommend that you post questions to a group on RG that Jeffrey R Brender started and is devoted to sharing amyloid protocols

https://www.researchgate.net/post/Amyloid_protocols_project

Article Specific Binding of Alzheimer’s Abeta Peptide Fibrils to Sin...

^ Hey, thanks for citing my work on ResearchGate! Do I get extra credibility points now and improve my score? I haven't really had time to read the whole thread in detail. I have deadlines coming up & life stuff to deal with. But what occurred to me is even if you can separate monomers out in aqueous solution, wouldn't they just snap back into dimers, multimers in fairly short order? This is particularly the case for amyloids like Abeta where you have to go through some hurdles to keep them from re-aggregating once converted to monomers (HFIP, DMSO, basic pH, etc...). Aliquots frozen at -80 C. Since amyloid fibrils grow through nucleated reactions, it wouldn't take much of the critical nuclei to have dramatic effects on the kinetics of the reaction (assuming that's a concern). So fun stuff to work with, indeed!

As far as the question in the OP.  Dimers would have the same IP as monomers if you think of the sequence only (assuming homodimers not heterodimers). From a structural point of view, the microscopic pKa of a given site can be perturbed by it's environment. For instance if salt-bridges or repulsive electrostatic interactions between like charges occur at the dimerization interface, I could see how the charges in the monomer could be quite different from the charges of each monomer in the dimer.

Sorry for the rushed answer. Gotta go!

Yes. I have separated mono/di/tri/tetraUbiquitin chains by Ion Exchange Chromatography. Longer and ordered assembly will presumably have stronger affinity for the resin, and will therefore, require higher salt for elution.