If i Lyse cell overexpressing a membrane protein with sonication in 20mM NaH2PO4 and 500mM Nacl, firstly can i load it as it is in sds page or do i have to add SDS loading buffer?
Either way , will it show up as a band in the SDS PAGE if it is insoluble?
If i separate soluble and insoluble, and resuspend the pellet in 1% Triton X-100 and leave it at RT for 1 hour would it posibly solubilize it? If so do i centrifuge again to separate or load the entire thing in SDS PAGE and again do i have to add SDS loading buffer or can i load it as it?
SDS is usually able to dissolve insoluble membrane proteins for electrophoresis. Triton X-100 may or may not be successful. Try dissolving the insoluble fraction directly in SDS-PAGE sample buffer.
Adam if SDS was able to dissolve insoluble proteins i'd see it when running the total lysate and not need to separate soluble from insoluble to begin with to check for its presence... Either my protein is not being overxpressed or some reason or is insoluble in SDS
The below Figure is taken from Senthil K. (2010) " Expression and single-step purification of mercury transporter (merT) from Cupriavidus metallidurans in E. coli"
It is the insoluble pellet treated with different detergents and is what is encouraging me to believe Triton X-100 could be better than others and that SDS will not solubilize the protein.
Fig. S 1 SDS-PAGE of various detergent solubilized merT samples Lane M, Molecular weight marker; Lane 1, sonicated cell pellet; Lane 2, over-night urea treated supernatant, Lane 3, over-night without detergents; Lane 4, 1% (w/v) SDS treated sample; Lane 5, 1% (v/v) Tween 80; Lane 6, 1% (v/v) Tween 20; Lane 7, 1% (v/v) Triton X-100; Lane 8, 0.5% (w/v) deoxycholic acid and Lane 9, 1% (w/v) N-lauryl sarcosine detergents in 20 mM Na2HPO4, 500 mM NaCl, pH 8.0
Can you do a Western blot to find out whether the protein is expressed in the total lysate? Experimenting with detergent extraction will be a waste of time if the protein isn't there.
When using Triton X-100, be careful about the quality of the detergent. Old bottles of this detergent can accumulate peroxides that destroy proteins. Use new detergent. If you can afford it, buy 10% Triton X-100 in sealed ampules under inert gas from Thermo-Fisher (Surfact-Amps-100). Store unused solution in the freezer after opening an ampule..
If i don't see a band in the gel, would western blotting it make a difference?
Thanks for the tip on Triton X-100, I've been using a bottle that's been sitting on the benchtop for years
If the only way you can see a band is by Western blot, then the expression level is probably too low to allow you to purify the protein to homogeneity, but it may still be possible to get an enriched prep that can be used for some investigations. And, it allows you to work on optimization of expression and solubility. If you see no band by Western, then the protein is not expressed at all, and you have to think about switching expression systems or constructs.
I recently read i can also verify by checking for inclusion bodies in induced cells vs non-induced by phase contrast microscopy... Do you think that's a good idea?
Western is a problem since i don't have any antibodies
Yes, it is possible to see inclusion bodies by microscopy. You can also partially purify them by centrifugation, and they can be dissolved in strong chaotropes (urea, guanidine HCl) for protein purification. However, the protein will be denatured and will have to be refolded. This can be a difficult process to optimize, so it should be considered as a last resort.
One thing to remember and consider is that membrane proteins are a small fraction of cell protein weight wise (though not diversity wise). Simply put the volume of the membrane is much smaller than the volume of the cytoplasm. Given this if you use total lysate your membrane protein might be hidden in the 'forest' of the soluble proteins. I would suggest separation of membranes from soluble by ultra centrifugation (can be done in small scale if you have access to such), or you can try one of the kits (can't recommend any specific one so just google "membrane protein enrichment kit bacteria").
You might also find these papers of interest:
Article High-level production of membrane proteins in E. coli BL21(D...
Article Revolutionizing membrane protein overexpression in bacteria
Yoram
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