What if you have Triton X in your buffer and you need to concentrate your protein sample? Because Triton has micelles of size about 90 kDa, one cannot use gel filtration chromatography, or filtration devices used for standard concentration. So how to get rid of it? I was thinking about adding another detergent like CHAPS, which could theoretically create mixed and thus smaller micelles which could be removed more easily.
If your protein wont aggregate (or precipitate) with lower amount of detergent, I would say try incubating the concentrated protein in Biobeads to remove some of the concentrated TX-100. The removal of TX-100 can be easily monitored by UV absorption or fluorescence as TX-100 does absorb around 280 nm wavelength. I have not come across the mixing detergent technique, so that sounds interesting as well. If this indeed decreases your micellar size, a good way to check would be using light scattering I suppose! I hope this help! Good luck!
yeah, I think I've already heard about those biobeads. What are they? Are they from BioRad or is it just coincidence in names?
They are from Bio-Rad, these are non polar polystyrene beads that allows adsorption of small micelles. However, please keep in mind that you will lose a portion of your proteins as well when incubating with Biobeads.
http://www.bio-rad.com/en-us/product/bio-beads-sm-2-adsorbents
This brings to my mind - in principle, hydrophobic chromatography could be used?
Could you clarify specifically the hydrophobic chromatographic experiment that you want to perform?
well, load it on octyl sepharose column and elute. I suppose the detergent will bind tightly, while proteins are mostly eluted with anything with higher ionic strength than water
It could work, but you might run into a problem of protein-detergent complex that would be hard to be removed. This is the tricky part of membrane protein-detergent nature.
I got another idea, dilute the sample to get concentration of the detergent below the CMC and then either dialyze or filter-centrifuge.
@Andrei - that looks good, I'm just afraid my boss would not buy it. But I'll keep that in mind. Thanks.
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