I have tried, through different methods using different kinds of detergents, as found in hundreds of literatures, to solubilize the water insoluble proteins completely in their native state. The insoluble proteins were solubilized in the buffer solutions containing the pepsin or trypsin enzyme in low pH, but proteins were denatured, and degraded.
I want to dissolve the complex water insoluble proteins completely if possible in their own native state in unaggregated forms in the solution. I would be appreciated if any one of experts in protein and solubilization give the reasonable answers according to their experiences. Thank you
Are you starting with inclusion bodies/precipitated protein, or with membranes containing the protein in its native state? In the first case, you will probably have to completely denature the protein with a strong chaotrope (urea or guanidine HCl), then refold it by gradually replacing the chaotrope with a suitable detergent. In the second case, you will have to find a detergent that will dissolve the membrane while keeping the protein in solution but not denaturing it.
Thank you Adam for your quick response and informative note. I appreciate. I am curious with the membrane proteins. I have been looking for the suitable detergents or any kind of buffers to solubilize the insoluble proteins in its native state. I have tried with the buffers containing SDS, SD, triton X-100, NP-40 with different concentration of various salt in different pH. No one combination dissolved the whole proteins. It is, I feel, difficult to solubilize all the proteins because of different in their complexities and compositions. Please share your experience if you have any, possibly useful to solubilize proteins.
First of all you cannot have membrane proteins in their native state as they are surrounded by a hydrophobic medium. Increase the concentration of surfactant and use mild surfantacts such as tween.
Thank you Omar for your note. I will consider your opinion during preparation of my protocol.
Every protein is different. There is not one single method that will work for all of them. The types of detergents that are usually used to solubilize proteins out of membranes without denaturing them are either nonionic or zwitterionic. Ionic detergents are usually too denaturing. Some commonly used detergents are n-octylglucoside, dodecylmaltoside, CHAPS, and sulfobetaines (e.g. Zwittergent 3-12 and 3-14). Stabilizers may be added, such as glycerol, sucrose or trehalose.
Hello Girdhari. Perhaps you could read how we solubilised our membrane protein. It may help, as many detergents were tested in a range of concentrations before finding one that was suitable. Best of luck.
Article Expression, solubilisation, and purification of a functional...
Thank you Barbara for the paper. It is very informative. As you clearly mentioned in the paper, maintaining the functional proteins would be the challenge.
As Adam mentioned above, the detergents you listed are not usually suitable for membrane protein purification, if you would like to keep their native fold.
DM, DDM, L-MNG, GDN are much more suitable as they are not as harsh as triton or NP-40 and have CMC values that will allow for purification and concentration.
Your question is too vague to give a detailed response though. What expression system? Full length? Fusion proteins .....lots of literature on membrane proteins - might want to look at the link below. It has a database of all membrane protein structures and you can read the papers for their protocols.
Thank you Brandon for your critical suggestion. Would you send me the link, you suggested me to go through? Thanks in advance.
http://blanco.biomol.uci.edu/mpstruc/
I attached it last time but for some reason it isn't there.
http://blanco.biomol.uci.edu/mpstruc/
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides