Hmmmm…your reply is another sign that I have no idea what I am doing….I’m new to dealing with proteins. If you can persevere, let me tell some back ground and how I arrived at things.
I am trying to put together an empty nanodisc which has two components, an MSP scaffold protein which encircles a phospholipid, DMPC. The idea is that you bung them together at a particular ratio along with biobeads, remove the detergents, once removed they form a disc. In that disc you can assemble trans-membrane proteins and hope to make them soluble. But for now I want to see if I can make an empty one.
Firstly, the MSP protein was purified via His and gel filtration to clean it up. I fed the sequence into ExPASy ProtParam which predicted my mass extinction co-efficient at 0.802. My A280 absorbance (nanodrop) readout was 1mg/ml.
I calculated absorbance/extinction co=efficient ie. 1.0 / 0.802=1.25mg/ml (g/L).
Then mg.ml/MW (g/mol)=Da, =M concentration? ie. 1.25mg.ml/33.57g.mol=37mM MSP protein?????
I might have done this wrong or used wrong terminology……
The second component is DMPC which came as a powder. On the jar is was labeled 33.89mg, FW677.93. I reconstituted 50mM using chloroform (33.89mg in 1 ml), dried it out with N and freeze dry, then re-suspended it in 100mM Sodium cholate (volume to volume).
My next step is to put them together incubating with biobeads, but the ratio must be 1:160 (MSP:DMPC) for it all to fit, then gel filtration max volume of 300ul. The protocol (Ritchie et al., (2009), Sligar lab) did not specify what type of ratio it was, I assumed molar.