Hello everyone,
How does one determine the concentration of a sample with multiple proteins? I.E. If you have a complex that is formed from 3 different proteins, how do you determine it's concentration using A280?
For accurate concentration calculation, I denature my proteins prior to measuring absorbance (extinction coefficient is calculated based of sequence). However, the extinction coefficient of the complex (all 3 proteins bound) is going to be different than the proteins individually. So I can't use the extinction coefficient calculated of the entire complex, but I also can't use the extinction coefficient of each protein individually.
I'm curious how this is done in general, since in papers, you often see multimeric complexes with given concentrations. How are those calculated if you have the problems above? Do you determine the extinction coefficient in the bound form (through another technique outside of calculating from the sequence), and then use A280 without denaturing the protein? I.E. Extinction coefficient and A280 now represent the folded protein rather than the unfolded protein in my case.
If you want accuracy, the ratio of absorbances of folded and unfolded complex equals (at the same concentración) the ratio of the corresponding extintion coeficients. And the ext. coef. of the unfolded complexe is the one you compute from the three sequences.
You TVneed yo know the stoiquiometry, of course.
If you know the stoichiometry of the proteins in the complex, then compute the extinction coefficient of the denatured protein on the basis of the sequence of all the subunits in their proper stoichiometry as if it were a single protein. For example, if the protein consists of 3 subunits with stoichiometry A1B1C2, then calculate the extinction coefficient of the sequence A-B-C-C. I think this is essentially the same suggestion as the one from Javier Sancho.
Hi there,
Just compute the full complex sequence as a fusion protein respecting the stoechiometry as suggested above. Make sure the sample is pure in terms of complex (ie. absence of any subcomplex or monomers).
Thank you for the suggestions; however my concern is you don't know much of each protein in the complex of 3 proteins you have. It's a 3:1:1 ratio, but what if you have 10%:10%:90% of these proteins? So if one protein contributes to 90% of the absorbance, and if you calculate the concentration based off the extinction coefficient of the entire thing, it'll be incorrect. These proteins are all similar molecular weight, so I can't determine how much of each I have via a gel.
https://sciencing.com/calculate-concentration-solution-different-concentrations-8680786.html
The content of the URL provided by Farshad Pazhoh is unrelated to the original question.
The situation is unusual, you say you have a 3:1:1 ratio but then you say it could be 10% 10% and 90%. It is not clear what you mean by "don't know much about the proteins". With such an uncertainty, it appears not to be crucial knowning the concentration with high accuracy since you don't know the ratio. I suggest you use Bradford, in our hands and for a large list of GLOBULAR proteins it never differs by more than 10% from A280
Gonzalo de Prat-Gay
What I was trying to say is the ratio of the proteins in the complex is 3:1:1; however that is the ratio when they are bound. You could have a proteins that are not bound, floating in solution as well.
E.G. Say you have 3 proteins A, B, and C that all bind to form an ABC complex in a 1:1:1 ratio. You want to determine the concentration of ABC you have in solution. If you go the route that has been recommended, you assume your absorbance is entirely due to the ABC complex. You read the absorbance, and get a very high output. Using the extinction coefficient of ABC, you have a lot of complex. However, what if the solution you have, is very little of ABC and is primarily protein A.
My confusion is:
How do you confirm how much of your complex you have. If you are doing titration experiments, you need to know the concentration of your complex, but what if you have very little complex and the absorbance reading you are obtaining is not from your complex, but another protein. Bradford reagent would come across the same issue.
Then your problem seems to be how much complex or in other words to what extent was the complex formed. Since you don't know the dissociation constant you don't know at which concentration it will be all complex. Accurate concentration measuremente won't help you for working out stoichiometry or the ratio free-bound of the tcomponents. I believe you should run SEC experiments and look at the amount of complex or free proteins. What you really need to know is the stoichiometry and the concentration at which you have saturated complex. Perhaps you are able to work out the stoichiometry of 2 of them and then titrate the third. Since there will be a 1/5-1/10 dilution, if the complex is weak, then you will not have full formation. If it is tight you will.
Something unbelievable effective and simple is running native agarose gels, of the proteins at different concentrations and/or ratios, you will see saturation when you see a bandshift and the free species start to increase. You can use whatever concentration you want (10 µM or more) and you can scan the bands and get a fairly good quantitative estimation.
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