My protein is scfv 25kda which precipitates after puirification through ni nta chromatography.
Protein precipitation is observed next day when protein is taken out from refrigerator.
Is there a way to resolublize the protein?
Equilibration buffer: 20mM tris, 150mM Nacl, pH8
Wash buffer: 20mM tris, 150mM nacl, 25mM imidazole pH8
Elution buffer: 20mM tris, 150mM Nacl, 250mM imidazole pH8
Here are 3 reasons why the protein may precipitate on storage:
- The concentration of the protein is higher than its solubility under the storage conditions. Storing the protein at lower concentration may be sufficient to solve the problem. If not, it may be sufficient to change the storage buffer, such as by raising or lowering the pH or the ionic strength, by including a nonionic or zwitterionic detergent. If the protein has exceeded its solubility, it may be possible to redissolve it by the means just mentioned.
- The protein may be unstable under the storage conditions, gradually becoming denatured. In addition to the changes to the storage buffer mentioned above, it may help to include a stabilizing additive. Some examples are a ligand such as a substrate, glycerol, PEG, sucrose and, my personal favorite, trehalose. If the protein has precipitated because it is denatured, you will not be able to redissolve it without going to great trouble to refold it.
- The protein has undergone proteolysis during storage, leading to an insoluble or unstable product. Contaminating proteases can sometimes be inhibited by addition of a protease inhibitor cocktail. Improving the purity of the protein through additional purification steps may get rid of contaminating proteases.
You should also consider storing the protein a different way, such as frozen in small aliquots (stored at -80oC or in liquid nitrogen), as an ammonium sulfate precipitate in the refrigerator, or in 50% glycerol at -20oC.
Excellent suggestions by Dr. Shapiro. I just want to add a little part for this, try to exchange the protein in a new buffer after elution, like 20mM tris, 500mM Nacl, no imidazole, in case 10 % glycerol and 1 mM EDTA, pH8 (if your protein like this pH) and store in higher volume (low concentration). If it doesn't work, you change the buffer and add some cocktail (as Dr. Shapiro has also mentioned), optimize pH to check your protein stability in various conditions. Good luck!
I study a protein which precipitates if the concentration of sodium chloride is less than 200 mM. So all my buffers have 400 to 500 mM sodium chloride. Your protein might be like this so just lyse and wash and elute your protein in high salt buffer. Also glycerol does wonders for keeping proteins soluble so you should be using 10 to 15 % v/v glycerol in your buffers. Glycerol is an antinucleating agent meaning that it stops proteins from aggregating together and coming out of solution as an amorphous precipitate or as crystals in most cases.
Thank you Adam, Somnath and Adron.
I will follow your suggestions and will let you know.
- Badges
- Science method