I am wondering if anyone can explain some apparent weight discrepancies between SDS-PAGE and Blue Native PAGE. I understand that the underlying principle is different in each, which may produce some differences due to hydrodynamic radii. However, for example, HIV envelope trimers run on SDS-PAGE at around 100-130 kDa (ours around 100 since they're truncated). However, when the trimer is run on a Blue Native PAGE, the apparent molecular weight is around 600-700 kda. This seems universal across many publications from different groups. Taking into consideration then the hydrodynamic radius, the Env trimer is actually very similar to the Thryoglobulin ladder standard (8.1 vs 8.6). Many groups have confirmed by DLS that this 600-700 kda band is in fact trimer.
Now, based on theoretical analysis and DLS experiments, the trimer should have a total weight (including glycans) of around 350 kDa. Why then, is the trimer showing up at almost double this expected weight on NATIVE PAGE, especially when it should be well-represented by the ladder standards? Glycoproteins will migrate slower, but really this much slower?
I am most concerned with this because I am working with other viral glycoproteins, and my lab does not have DLS or another powerful method to accurately determine the weight. For RSV F trimers, for example, the SDS PAGE shows around 70 kDa for monomer, while the trimer is around 400 on Native. Given this, the expected trimer weight (~ 210 kDa) and the apparent Native PAGE weight (~400 kDa) are roughly similar in proportion to the HIV trimer. I am, however, hesitant to believe it is well formed trimers, and not, say, a dimer of trimer (aggregate).
Does anyone have any insight or advice for assessing the oligomeric form of these proteins, and possibly an explanation for this large weight discrepancy for the two types of electrophoresis?
Native gels are not adapted to determine the Mw of proteins or protein complexes since the migration depends not only on the size, but also on the shape and on the charge of the species. The effect of charge can be taken into account by running Ferguson plots with gels run at different percentages of acrylamide, but the shape will still affect migration. Your best bet is probably analytical ultracentrifugation.
If PAGE is unable to resolve the Mr of the HIV protein, possibly a trimer, you may give a try with gel exclusion chromatography on a Sepharose 2B-CL column using a mobile phase without SDS and a reducing agent. You just as in case of PAGE, shall need a mix of reference proteins to calibrate a reference curve between Ve and the A280 values.
In SDS-PAGE, the ambiguity in ionic mobility shall likely to happen because of bigger size of the protein under investigation because of shape and size(s) of the protein molecules.
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