I have expressed HBsAg-S together with a small tag in HEK cells. The particles self-assemble after expression. I want to reduce my VLP now and test its monomers.
How can I do this?
Preferably I would like to do this without the use of detergent, because the monomers will be used in ELISpot assay later on (Detergent = ELISpot membrane leakage).
This depends on the mode of multimerisation.
If it is from hydrophobic interactions then detergents will be necessary. Perhaps you can use a detergent with a very low CMC and it will be diluted to negligible levels when you add the protein to the ELIspot. You can also try non-detergent sulfobetaines (NDSB) that are often used to prevent hydrophobic aggregation of proteins.
If multimer formation is via charge interactions, then addition of salt (NaCl from 50-500 mM) or pH shift to 2 points above or below pI of the protein can help.
If it is by disulphide bridge formation, you can maintain a reducing environment by treatment with DTT or TCEP and storage in degassed buffers.
If a combination of these factors is responsible for the self-assembly multimerisation then you have a screening experiment to do! And your buffer controls will be critical in the ELIspot.
I hope this helps.
Thanks alot for the very detailed answer Deepan!
As far as I know, the HBsAg-S VLP is held up by hydrophobic interactions. Therefore I have to use detergents. It is possible thought that I can dilute the sample to a grade where no problems should appear in the ELISpot.
I will give it a go.
Thanks for the rapid response
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