Hello, I am working with membrane proteins expressed in Sf9 cells. My protein is fused to mRuby2 red fluorescent protein for FSEC purposes, but it seems that it doesn't extract efficiently from the membrane with any of the detergents tested so far (doesn't extract at all with non anionic detergents such as DDM, extracts/solubilizes a little with zwitterionic Fos cholines). I work with above CMC values for all detergents. The protein is clearly localized in the membrane since the purified insect cell membranes are pink after visual inspection. Does anybody have similar experience and could give me some advice on how to proceed? I am working with the full length protein, harboring the extracellular, transmembrane and cytoplasmic domains. I haven't tried multiple constructs with truncated regions yet, since I would prefer to work with the full length wild type protein rather than with truncated constructs. Moreover, my final purpose is not to crystallize the protein, but rather produce it in sufficient amounts for biochemical and biophysical characterization.
Dear Ioanna,
the CMC depends on the ionic strength of the buffer. If you work with high salt concentrations, you might have to increase the detergent concentration significantly. One detergent that is very efficient for the solubilisation of lipids is sodium cholate. But I`m not sure how stable or well folded your protein would be in those micelles. Maybe you can perform an exchange to a milder detergent after the extraction.
Cheers, Philipp
Thermo Sicentific has a wery nice kit for this kind of experiments and it works well
https://www.thermofisher.com/ch/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/efficient-mammalian-membrane-protein-extraction.html
Greatings, Natasha
Hi all,
I used DDM at 2%. My solubilization buffer is 50 mM Tris and 150 mM NaCl, so the physiological salt concentration. I will try to use sodium cholate, I have read about its use either alone or at 0.01% in combination with other detergents. I will also try to solubilize it in SDS, as a positive control, to see how much protein I can extract. I've also read about solubilization in SDS and subsequent refolding in detergent/lipids. Do you have any protocol that works to suggest?
Thanks to all
You can read our paper at
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111341
Regards
Jarek
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