Dear all,
I used both mass photometry and SEC-MALS to define the mass of my recombinant protein. Based on the amino acid sequence only, it should be 92 kDa. However, it holds different glycan chains that increase its molecular weight. When I tried to measure the mass of my protein, I obtained all different values: 118 and 139 kDa with mass photometry (the two values were for my recombinant protein in solution in two different buffers) and 86 kDa with SEC-MALS. Why such a difference? What could be the reason? Why SEC-MALS measured a MW that was less than the basic MW for my protein?
Hi,
I've not worked with SEC-MALS specifically, so take this with a grain of salt. Anyway, light scattering experiments depend on several assumptions made on your molecule, mainly, that they have similar characteristics to the standards that you use to judge the size. For example, if your protein is more compact than the standard, it could appear smaller to the machine. The light scattering doesn't give information on the mass or density of your protein, only on the area (or perhaps volume?) that it takes up.
Hello Tany Nino
concerning your specific question why SEC-MALS measured a MW less than the expected MW for your protein:
- check the concentration determination, are you using UV? correct absorption coefficient? Are you using RI? correct dn/dc?
- check the dn/dc for the wave length of your system, it enters as (dn/dc)^2 so a relatively small error can lead to a few percent off
- check the calibration / toluene of your MALS system
- is the column/buffer suitable, if anything is stuck to column or any pre-filter then the true concentration is lower than the calculated/expected concentration
In essence your experiment indicates you are getting less light scattering signal than you expected, so search for potential influences that could lead to that.
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