Does anyone have an idea of a good lysis buffer detergent for membrane protein purification? I am working on purifying a single-span membrane protein, and I have tried SDS, IGEPAL, Triton, and DDM. These did not give desired protein peaks in size exclusion purification.
If the membrane span isn't important to your study, consider expressing the protein without it as a soluble protein. It will make your work much easier.
With size exclusion chromatography, you have to consider the effect of the detergent micelles, which add to the size of the protein. Also, if the amount of detergent is not high enough so that every protein molecule has a micelle to itself, you could end up with more than one protein molecule sharing the same micelle and appearing to be an oligomer. Furthermore, insufficient detergent in the buffer could result in aggregation. The SEC column should also be equilibrated with the detergent to prevent it from being removed by the chromatography.
Thanks Dr. Shapiro, the transmembrane domain is important to my study.
Hi,
Sometime adding 10% glycerol can help. Although I have had proteins with both negative and positive effect of having glycerol, so you just need to test it out.
How do you lyse your cells? From my experience, I get better yield when using cell disruptor compared to sonicator.
After you lyse the cells, keep the solution in coldroom rotating for an hour(1h-3h-5h had same effect so). Previously, only FosCholin-12 was only detergent I was able to solubilize my protein, but after lysing and keeping in the coldroom for an hour, my protein is soluble in every detergent I have tried (with better or worse yield of course). Some 20x CMC of the detergent is a good start.
Like Adam B Shapiro said, keep the detergent in the SEC-buffer when you run your protein through it. At this step, less detergent it required. This is another step of optimizing, and the required range can be from 0x CMC to 10x CMC. This is also good step if you are thinking of switching the detergent to another.
When you say it doesnt give desired protein peak, do you mean the yield or retention volume? If you have protien-detegent complex, the protein behaves as if it would be larger -> earlier retention.
Eq. My monomeric protien is 17kDa, but purified with DDM or C12E9, it behaves as if it would be 100kDa. Running this is SLS and doing protein conjugate analysis, I found out that there is roughly 80kDa+ detergent micelle around the protein. Different detergents can obviously have larger or smaller impact on the overall size (and shape) of the micelle.
Hope this helps in any way!
Thanks Mikko. I use two lysis buffers and do have 10% glycerol in the first.
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