Hi. A quick and dirty way of approaching this question is to run each peak again on the same gel filtration column. If it is an oligomer then it should be in equilibrium with the monomer and vice versa. If it is an aggregate then the peaks will probably not shift.

Hi. A quick and dirty way of approaching this question is to run each peak again on the same gel filtration column. If it is an oligomer then it should be in equilibrium with the monomer and vice versa. If it is an aggregate then the peaks will probably not shift.

You might like to calibrate your column using reference proteins (BSA, Lysozme, there are also commercially available kits) to get an idea which size the complexes are and if they could correspond to an oligomeric form. Otherwise, if you can separate both peaks, analyse them in non-reducing SDS-PAGE or native PAGE to see if this high molecular species is aggregated material or migrates as defined band as typical for an naturally occuring oligomer.

@Steingrimur Stefansson : when i pool the peak the protein is very much diluted so i dont know if i re-run each peak whether i ll get any sharp peak!!

@Michael Mühle: the column is already calibrated.

after gel filtration run, i usually check on the SDS-PAGE and then only pool for further process. i tried native PAGE along with BSA as marker, but my protein doesnt enter the resolving gel and BSA does. i tried with and w/o stacking gel also in-spite of that also the protein doesn't enter the resolving gel.

does the aggregation of the protein is concentration dependent ?

Hi Ambika. If you have a good assay for your enzyme then you really don't need to see a sharp peak to know where your enzyme comes out.

Is the high MW peak active? If so, you can rerun that fraction and see the lower MW activity appear, if the HMW peak is oligomers. If that peak is inactive, then it is more likely aggregates that will not yield an active enzyme.

Protein aggregation does not have to be concentration dependant, although it is more observable in concentrated samples. In general, protein aggregation occurs when the native protein structure is less energetically favorable than the unfolded protein structure. The reason for an energetically unfavorable protein structure is mostly dependent on its interaction with its solvent.

If the protein does not enter the gel with native PAGE you very likely either have an aggregate (more likely) or an a defined oligomer (less likely). What is the MW of the monomeric protein?

I'd check how defined (i.e. broad and shaped) the band on GF is. It would be best to use some sort of analytical FPLC or HPLC.

@Steingrimur Stefansson: Thank you, I appreciate the suggestions. Right now I'm into checking the activity of both the peaks. Re-running in GF is not possible because of lack of facility (FPLC/HPLC), i generally use conventional gravity gel filtration column. I do see some activity in the HMW peak but not exactly the same activity as like LMW peak. Thank you very much.

@Jeffrey Brender: I ran native PAGE for both the peaks, none entered the gel. I expected atleast the LMW peak should enter the gel!!! MW 73kDa.

@Tomáš Hluska: Hi, both the bands were sharp when i loaded 2ml, and broad when 10ml was loaded in a 120ml bed volume column.

If you have access to a dynamic light scattering instrument, you can measure the particle size distributions of your two fractions to determine whether the higher molecular weight fraction is a well-defined oligomer or a heterogeneous aggregate. A SEC-MALS system (size-exclusion chromatography-multiangle light scatter) is even better, since it combines separation with mass measurement.

the peak you are getting after the void volume is definitely LMW protein and the peak you are getting near by the void volume of the column is the high molecular wt compound. i want to know that which gel you are using ? its either sepharose 6B or Sephadex-200 OR any other . if your compound MW is likely to be 200 kd or less i would suggest you to use sepharose 6B. i am using frequently the same column what you mentioned for fish protein separation.