Greetings everyone! I'm trying to find out one of the protein's oligomeric nature. I used three different approaches to identify the oligomeric status. I found the range of oligomerization, from trimer to 12mer, on the BN page. Only a 9mer was observed in size exclusion chromatography, whereas dimer, trimer, and tetramers were observed in SEC MALS. Now I'm thinking of how to sepculate this in the discussion and result sections.I would really appreciate anyone who can help me with this problem. Thank you.
The observed distribution of oligomer sizes could depend on the concentration of the protein during the analysis, if the monomers and oligomers are in equilibrium. The separation methods are not equilibrium methods, since the concentration changes during the separation, so the method used can affect the results. The environmental conditions (e.g. pH and ionic strength, and the presence of Coomassie Blue dye) and temperature can also affect the equilibrium. Size exclusion chromatography without MALS probably does not have sufficient resolution to distinguish the various oligomers and gives a result reflecting the most common oligomer (i.e. the peak).
I would say that the results indicate that there is an equilibrium established between monomers and a range of oligomers over the range of protein concentrations used and the range of conditions used.
You may be able to learn more about this equilibrium by conducting these and other experiments with varying protein concentrations.
I'd try to determine the enzyme activity of the various oligomeric states, for example in a zymogram. Analytical ultracentrifugation can also do that, provided you can follow the reaction spectrophotometrically.
Some enzymes (morpheins, proteins that morph) have low activity in one oligomerisation state and high in another,
Engelbert Buxbaum The protein is a structural adapter protein not an enzyme.
Adam B Shapiro Thank you sir for your valauble suggestions.I tried with different concentration of proteins.In lower concentration i got nothing in BN page but i got lower absorbance peak in the Size exclusion chromatography corrosponding to 9mer.
Hello Abrar Hussain Qadri ,
if you have access to a Dynamic Light Scattering system and your sample is relatively well-behaved (i.e. not any large aggregates) then you may be able to get a little more insight into the situation with DLS. The width of the peak in DLS can give a hint on the presence of oligomeric states, without any column interactions. Here are a few potential blogs that might be of interest for you
- https://www.materials-talks.com/faq-molecular-weight-estimate-from-dls/
- https://www.materials-talks.com/zetasizer-sensitivity-for-protein-dls/
- https://www.materials-talks.com/polydispersity-what-does-it-mean-for-dls-and-chromatography/
- https://www.materials-talks.com/key-protein-features-in-the-zetasizer/
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