I have a single purified sample of trimer, dimer and monomer proteins purified through affinity chromatography.
Is there a way to find concentrations of each protein in a sample?
If total concentration is 5mg/L then how much is trimer, dimer and monomer in 5mg of total protein?
If the monomer, dimer and trimer are not in rapid equilibrium, so that they can be separated from each other, you can quantify them using analytical-scale gel filtration chromatography with UV detection, or by analytical ultracentrifugation, or by native gel electrophoresis.
Dear Adam,
I am using native page (75kDa, 50kDa, 25kda). Are you suggesting me to use densitometry technique to quantify them?
If you have well-resolved, stained bands, you can quantify them by densitometry, keeping in mind that the amount of bound stain is proportional to the amount (in µg) of the protein in the band, not the number of moles.
Low angle laser light scattering (LALLS) can determine the concentration of monomer, dimer and trimer even if they are in rapid equilibrium and hence cannot be separated. You will probably have to cooperate with someone, who not only has that equipment, but also experience with the (non-trivial) evaluation of the results.
According to the manufacturers' website (https://www.malvernpanalytical.com/en/support/product-support/zetasizer-range) the zetasizer performs dynamic light scattering (DLS), that is, it measures the noise component of scattered light caused by the Brownian motion of the particles, and hence their translational diffusion coefficient Dtrans.
Once the size of the particle becomes larger than λ/10, photons scattered on different parts of the particle interfere, scattering intensity then depends on the scattering angle. This can be used to not only determine Dtrans, but also the radius of gyration rG, that is, roughly, the shape of the particle.
This complication can be circumvented by measuring at low angle. Ideal would be 0°, but then the measurement beam would interfere. However, the angle should be < 10°. See doi:10.1016/0142-9418(83)90013-2 for more details.
Hello Saleha,
while LALLS is not part of the Zetasizer, it may be possible to combine the info from dynamic and static light scattering if you can assume that there is nothing else in your sample than monomer, dimer and trimer. For that you would need to know/measure the hydrodynamic size of your monomer, dimer, trimer plus the sample. Then also measure the molar mass of your sample, and if everything is perfect there should be only one solution that exlpains the DLS and the SLS result.
You might find this of interest Article Dynamic Light Scattering as a Relative Tool for Assessing th...
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