Two issues about membrane protein analysis want to be addressed here:
1. after sodium carbonate extraction, will you collect the supernatant for protein precipitation? if yes, how did you do that (TCA doesnot work, i tried) and why? i am concerning: for peripheral proteins, it can be extracted by alkaline solution (pH8-12). Does it mean they will dissolve into the alkaline solution or it can be spin down by ultra-centrifuge (100,000g)? I read many protocols and most are just discard the supernatant, but there do have one collecting the supernatant for further steps.
2. insoluble particles after methanol-chloroform precipitation. i used this method to precipitate my membrane proteins, but the pellets cannot fully dissolved in Tris-4% SDS. I was not overdrying it for sure. And the insoluble particles were solid and hard, feeling not like proteins. but if it is not proteins, what should they be? i tried to use more buffer and heat the solutions, but they are still insoluble.
Thanks.
Regarding 2), what is the composition of the solution out of which you are trying to precipitate membrane proteins? Many salts precipitate if present at high concentrations during MetOH/CHCL3 precipitation (GuHCl, for instance, needs to be diluted below 2 M). Also, what is your downstream application? If the pellet turns out to be just proteins and you intend to analyze the samples by SDS-PAGE, try resuspending into 8 M urea + 5 mM DTT and see if it makes a difference.
Thanks Alejandro.
The solution is just MiliQ water. I resuspend the membrane pellets after sodium carbonate extraction into H2O and use it for the precipitation. So, theoretically, it shouldnot contain many salts. I use Tris-4% SDS to dissolve the precipitates, as SDS is required to dissolve membrane proteins. I tried to add DTT as well, but still some insoluble particles observed.
And what I am most confusing about is the insoluble particles are solid and hard, just not feeling like proteins but more likely to be others (maybe lipids, ... ?). Not sure what it can be if it is not proteins.
Hi
Have you tried to use a different detergent instead of SDS, for example, DM (n-Dodecyl β-D-maltoside)?
Hi,
Yes, SDS is a strong detergent, I used DM for solubilization of hydrophobic cytochrome b6 and its work much better. DM is very useful for stabilization of membrane protein, in contrast to the SDS which destabilizes a-helices, also SDS is precipitated by potassium ions.
In my opinion should be compatible, I used DM in protein digestion with different proteases. DM works fine with DTT.
I have also faced the same thing! The insoluble thing is like plastic! What should it be, I used RIPA lysis buffer, DTT, and IAA, then methanol/ chloroform ppt.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts