Dear Rafael, What final concentration of DMPC are you trying to achieve? Or are you more concerned with a ratio?
For example, 20 mg/ml DMPC is nicely soluble in 50-100 mM sodium cholate (TRIS 7.8, 20-100 mM NaCl), and for preparation of micelles one can then suck the cholate out with bio-beads... the result will be liposomes of varying size. Heating to ~30C is helpful, whereas high heat may be problematic. Also you wrote 200 *nM* (nanomolar!) cholate - I suspect you may have meant something higher than that? Cholate is often used at 100 or even 200 mM millimolar concentration... guessing it's a typo. For nanodisc preparation, the fairly standard process involves adding membrane protein into MSP pre-mixed with a 'lipid buffer' containing 100 mM Cholate as well as the requisite amount of DMPC (65-100 molar fold over MSP, so about 1000-2000 fold over the molar amount of the membrane protein). I've not experimented too much with this method in the absence of at least MSP but generally speaking Cholate should help DMPC to go into 'solution' (it's not quite a real solution what with being mixed micelles and such). One other thing you may want to check is the purity of your reagents, especially DMPC - I've encountered batch issues with some of the ahem... *more affordable* suppliers in the past. Sometimes being a cheapskate is a bad thing. Artem - Cosmic Cats approve of this message On Mon, Dec 2, 2024 at 11:56 AM Rafael Marques <[email protected]> wrote: > Hi folks, > > I have been trying to prepare a lipid mixture for nanodiscs but I have > been failing miserably to solubilize > 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in my buffer. > > What I did so far was preparing 100 mM DMPC in chloroform, dry it out in a > round bottom flask and ressuspend the lipid in 20 mM Tris/HCl pH 7.4, 100 > mM NaCl. 0.5 EDTA and 200 nM Na Cholate. We treated the mixture at hot > temperature (60 to 100 Celsius) and sonicated it for long periods of time > with no success. The protocol descbribed in the literature says the > solution shoud be clean, but this is not what I am getting. I wonder if > someone experienced this issue before and could shed light in how it was > solved. > > Best wishes > > > > ______________________________________________________ > > Rafael Marques da Silva > > PhD Student – Structural Biology > > University of Leicester > > Mestrando em Física Biomolecular > Universidade de São Paulo > > Bacharel em Ciências Biológicas > Universidade Federal de São Carlos > > phone: +55 16 99766-0021 > > * "A sorte acompanha uma mente bem treinada"* > *________________________________________________* > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
