I want to determine the percentage of unfolded, native and aggregated form of my protein in a sample.
As some researchers in this site have suggested, I want to use sec chromatography. The molecular weight of my protein is 20,000 and therefore the aggregated form has a minimum molecular weight of 40,000 ( because the aggregated form is consisted of at least 2 monomers). Given the picture that i have attached, which kind of gel is appropriate?
I think sephadex g-50 is the best, am i right?
For a 20 kDa protein I would use a superdex 75 or superdex 200. If you want to detect large aggregates and estimate a size definitely the superdex 200. I would probably go for a 24 ml column but that of course depends on the amount of protein you have!
You may also want to consider static and/or dynamic light scatterign experiments in order to achieve more accurate results than from SEC alone. Hope this helps!
Mr Moll, would you please tell me what is your reason for choosing this?
I mean that, for instance, why don't you choose sephadex g-100?
Hi Reza,
personally I guess I would go for the superdex columns because I can easily get prepacked columns from different companies and I know they will usually do the job in this molecular weight range. I do not have any experience with sephadex media apart from desalting purified proteins. However, the molecular weight range seems fine for sephadex G-100 as well.
Superdex has a larger MW range on the high end, which will be useful if you are actually trying to quantify which type of aggregates you have
If you are only trying to isolate the monomer sephadex G75 or G100 would work
Sepharose 6B is a cheaper alternative to Superdex 200 (about 1/5 the cost of Superdex)
The protein you are working on is Interferon-β-1b correct? If so, you may want to look here http://pubs.acs.org/doi/full/10.1021/bc050322y
I just want to know the percentage of unfolded, native and aggregated form of my protein in a sample that is taken during a refolding process ( isolating this forms of protein from each other). Would you think that sephadex g-75 or g-100 are appropriate for this purpose?
Yes, my protein is exactly Interferon-β-1b and thank you for your help.
@ Reza "I just want to know the percentage of unfolded, native and aggregated form of my protein in a sample that is taken during a refolding process"
1. Centrifuge the protein. This will separate large unsoluble aggrregates from monomer and small oligomers. Measure the concentration of each fraction. Soluble +Insoluble should = Total
2. Use sepharose 6B to determine the relative concentration of species in the soluble fraction.
3. Folded vs. Unfolded: It is far easier to measure DeltaG approximately than to directly measure the concentration of folded/unfolded. Perform a chemical denaturation with urea using CD or SyproOrange fluorescence as measure for the unfolded state. Extrapolate DeltaG unfolding to infinite urea dilution (DeltaG-H2O). The percent unfolded can be calculated from this value. Directly measuring the concentration of unfolded would require some difficult experiments like Relaxation Dispersion NMR.
Dear Jeffrey, thank you for your perfect guidance but i have a question :
We know that there is difference between size of aggregates and monomers, so they can be separated by sec. Someone has mentioned in one of my last questions that native and unfolded forms also have differences in their gel filtration retention time.
Can sepharose 6B alone determine the percentage of each form?
Theoretically yes but it will only work in the unusual case that protein folding/refolding is slower than the elution from the column. In most cases you will just get the average of the two forms. Also i would assume the unfolded fraction is small which will make detecting it on the column difficult.
http://pubs.acs.org/doi/abs/10.1021/bi00211a042
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