Hello, I am new to membrane protein world. I have purified a membrane protein (with His-tag, no GFP) complex from E.coli membrane using one particular detergent. Now, I need to exchange this detergent with some others in order to screen it for better stability, homogeneity, and/or crystallization of the protein. I came to know some ways to exchange detergent for ex. gel-filtration, dialysis, but these methods are not very appropriate for me as some detergents are very very expensive to use it in more amounts in larger volume buffer.
Hi there,
To limit volume consumption I would go for immobilizing protein on NiNTA resin and exchange buffer. The problem being how to actually measure the detergent exchange efficiency unless you have in hand a specific detection for the detergent you want to eliminate.
I tried buffer change of my membrane proteins with the Millipore Amicon filters (molecular sieves). Tried it with dodecyl maltoside, which is nearly impossible to get rid of via dialysis. I filtered and diluted my protein solution 4 times, still a tiny amount of the detergent present.
In our lab we concentrate protein and run gel-filtration FPLC, for example SD200. This way you can exchange detergent, further purify your protein and estimate oligomerization state: monomers, dimers, "whatevermers", aggregation, or (most often) all together.
Hi Shiv,
It depends on couple of factors. Depending up the CMC detergent exchange method varies from on column exchange during purification, dialysis, dilution or more complicated methods such as gel filtration. If I was you, I would first try washing protein bound Ni resin with new detergent (50-100 column volume).
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques