My task is incorporation a RAGE protein into nanodiscs. I try to do it but results were not successful. After reconstitution nanodiscs I have a nice nanodiscs but it contains a small amount of target protein. I performed it twice and I have the same results. For nanodiscs I use DPPC lipid. Ratio protein / MSP - 1/5. Ratio MSP/lipid - 1/90.
May be I use wrong lipid?
May be protein degradate during removing sodium cholate, because I did it at 37 ºC (for DPPC).
I would be grateful for any help!
the strong band in the middle of the gel is MSP? The weak band on top (around double the size of MSP) is RAGE?
It seems your starting concentration of RAGE is already quite low. I would try to get it to a 1:2 ratio. load a sample of the mix before running it on the column. Just to get an idea of the ratio.
How do you remove the detergent?
Yes, the strong band MSP and weak band it is RAGE. I remove detergent using biobeads biorad another experiment i performed dialysis.
so your experiment actually might have worked. there is a band of your protein along with the MSP in the main peak. since your starting concentration is low you cannot expect more than that. If your protein does not have a larger cytoplasmic domain, the hydrodynamic radius of the Nanodisc won't be affected very much. Therefore filled discs would elute from the column along with empty discs.
Run a sample of empty discs on the column and try to get a better ratio of membrane protein to msp. like 1:1 or 1:2.
Furthermore you have quite some proteolytic digestion in your msp. you might have missed using inhibitors.
good luck
Hello Alexander,
to give appropriate advice you should provide more information such as protein source and molecular weigh, expression system, or the time exposed to detergent. Do you have to use DPPC for any particular reason? If not, I'd use POPC (Tm = -2 degree) and would work at 4 degree or RT if that's not a problem for your receptor. Your excess of MSP over protein is quite low, so if you have enough MSP available you should increase it. The higher the concentration of MSP the more you push the equilibrium towards the formation of the discs. Do you have any activity assay for your protein (radioligand binding, MST...)? If you do, you should check that your receptor is active after being at 37 degree.
Why should I increase MSP? I have A lot of nanodiscs but without target protein. It seems to me formation of nanodiscs - is correct. but they without protein. I have only DPPC.
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