Hi
I'm purifying some mutants of the protein I study. The wild type protein exists as a monomer and is 28kDa.
I have two mutants (same protein, same number of amino acids but with 8 amino acid substitutions at defined positions), one of the mutants (mutant 1) analysed using size exclusion chromatography with multi-angle static light scattering (SEC-MALS) and its MW was shown to be 33kDa and has an oligomerization state of 1.2. The other mutant, mutant 2, also measured by SEC-MALS was 59kDa with an oligomerization state of 2.2.
For the wild type to measure the concentration I've just been using the MW (28kDa) and extinction coefficient (calculated by entering the sequence into online software ProtParam) and using a NanoDrop measuring absorbance at 280. This gives the concentration in mg/ml which I then convert to molar concentration.
For the mutants I want to measure their concentration the same way - measuring A280 on the NanoDrop using the mutants MW and extinction coefficient and calculating molarity from mg/ml. I'm not sure if this is an obvious/stupid question but what MW weight and extinction coefficient would you use for the mutants on the NanoDrop? E.g. For example mutant 2 molecular weight (MW) of the protein based on its amino acids (AA) composition is predicted to be 28kDa, but SEC-MALS shows it is 59kDa as the protein forms an oligomer.
My instant is to use 59kDa and the computed extinction coefficient predicted from the AA composition - is this correct?
Thanks in advance!
I would calculate the concentration of monomer based on the UV method using the calculated extinction coefficient and monomer molecular weight, because it is performed under denaturing conditions in 6 M guanidine-HCl (or at least it should be if you want it to be accurate), so that the protein will be monomeric.
Afterwards, you can calculate the concentration of oligomer based on the SEC-MALS data. For example, if the SEC-MALS result is that the protein is a dimer, the dimer molar concentration is one-half the monomer concentration.
Since a protein can't be in a non-integer oligomeric state, the non-integer values from SEC-MALS indicate a mixture and the non-integer numbers are averages. However, I tend to think that numbers close to integer values are just the result of a small systematic error, and you should use the closest integer value.
Hi Adam B Shapiro
Thanks for your reply very helpful - so if I’ve understood you properly - it is incorrect to use the SEC-MALS molecular weight when measuring UV 280 absorbance to determine protein concentration of the mutants as the extinction coefficient is based on the protein being denatured. Rather I should use the predicted Mw just based on the amino acid composition.
I agree the deviation from whole numbers points to an oligomer mixture. I want to add these recombinantly purified mutants to permeabilised cells to observe their localisation (they are fluorescent proteins) so knowing concentration accurately is important for these experiments.
So for example for mutant 2 whose Mw just based on amino acid composition is 28kDa - if A280 on the nanodrop gives a concentration of 10 mg/ml which would be 0.35mM, the correct concentration of protein is 0.17mM? (as the oligomerizaiton state is 2 taken the closest integer value). I should say the ability of these mutants to form oligomers is because the mutations increase their aggregation propensities.
Am I correct in this? Is the above way accurate or would something like the Bradford assay be better?
In addition to Dr Adam, I would say that you should use similar technique and parameters from the same method from scratch for measuring the concentration of the mutant and the wild type.
If the SEC-MALS molar mass is used on nano drop for the mutant while Protp-predicted mass used for the wild type, it will be inconsistent since the two methods gave obviously different mass (and concentration). My take is, obtain the molar mass of the wildtype also with SEC-MALS (you can compare this with the predicted molar mass), to:
1. See if the prediction is correct (may differ due to aggregation).
2. To rely on the SEC-MALS data for both mutant and wildtype for nanodrop determination of the concentration, compare also with Bradford.
Kayode Adeyemi hi, thanks yes I am also using the SEC-MALS data for the Mw for the wildtype concentration measurements
The wild type is a monomer, but it’s about 2kDa larger than just the predicted Protp-predicted Mw based on AA sequence (predicted Mw 26kda, SEC-MALS Mw 28kDa) its only the mutants that are oligomers
- it’s just the extinction coefficients for both the wildtype and mutants predicted from ProtP are being used for the nanodrop, Mw values for measuring concentrations for both wildtype and mutants are from SEC-MALS
Daniel Solomon The concentration of mutant 2 dimers would be 0.17 mM. The concentration of monomers would be 0.35 mM.
It is possible that the dimers would dissociate when diluted if they are in equilibrium with the monomers at relevant concentrations. You might be able to find out using SEC-MALS if the sensitivity is sufficient for lower protein concentrations.
If the protein is highly pure (no contaminants with strong absorbance at 280 nm) and you make the absorbance measurement in 6 M guanidine, the 280 nm measurement of protein concentration should be pretty accurate. It's not a bad idea to also try other protein assay methods (e.g. Bradford, BCA, amino acid analysis) if an accurate knowledge of the protein concentration is important for the experiment. For methods that require comparison to a standard (Bradford, BCA), choosing a standard that is representative of your protein in terms of amino acid composition is important for getting an accurate measurement.
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