The power point attached shows the GFC data. IT was run on an Akta 25 pure using the sephacryl s-100 column. the elution volumes around 50mL is stating there are trimers. However, monomer formation is expected to happen at low pH.
Not able to open the attachment. Firstly, are you able to spike the sample with MW standards as internal controls? If the peak is broad, unlike the standards, then there could be interaction with the resin polymer and/or the protein subunit(s) is partially denatured. Do you get total recovery of the injected protein? Would re-injection of the trimeric peak still yield a trimer? Assuming non-covalent trimerization, does SDS-PAGE, reduced versus non-reduced, provide any orthogonal verification of the monomeric states of alpha and beta chains?
If there is trimer then it seems the a and/or b subunits could have some degree of denaturation since usually the ab dimer is in equilibrium with the tetramer at physiological pH with some slight changes in the heme at muscle physiological acidity. I've heard of adult Hb monomers, dimers, tetramers but not of trimers (maybe for myoglobin and I'm not sure about fetal Hb). Usually Sephacryl is for large scale operatons and therefore is made of dextran cross linked with methylene bis-acrylamide while Superdex is cross-linked agarose with dextran bonded phase which might provide less resin interaction.
The elution volume of a protein depends on its mass, but also on its shape, with less globular proteins tending to elute earlier than their mass would suggest. To check whether the eluted protein is really a monomer or a trimer, try using dynamic light scattering (DLS) or better yet size exclusion chromatography coupled with a multiangle light scattering detector (SEC-MALS).
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