Hi all,
yes, it was a typo. I have twice the amount of Na cholate than DMPC, in this
case 200 mM.
Best wishes
PS: I will answer individually to all of you. Thanks in advance for your help.
______________________________________________________
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"
________________________________________________
________________________________
De: Artem Evdokimov <[email protected]>
Enviado: segunda-feira, 2 de dezembro de 2024 17:17
Para: Rafael Marques <[email protected]>
Cc: [email protected] <[email protected]>
Assunto: Re: [ccp4bb] [OFF-TOPIC] Dissolving DMPC/ Na Cholate in buffer for
nanodisc assembly
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]<mailto:[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"
________________________________________________
________________________________
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