I have isolated the Triton insoluble protein aggregates from the cell culture media and blood serum. I got the pellet (insoluble protein) in the final stage of the isolation process and it’s not dissolving in the PBS, and I have used 8M urea to dissolve the protein aggregates but the 8M obviously denature the protein. I would like to solubilize the protein without denaturing them. Please suggest any protocols which can resolve this problem also I wonder whether the refolding of the denatured protein retain their native characteristics?
What do you plan to do with the Triton-insoluble proteins, so that you need to avoid denaturation?
You might try a stronger detergent, such as a zwitterionic detergent (e.g. sodium cholate, CHAPS, CHAPSO, Zwittergent 3-12 or 3-14).
Refolding is typically the option used. This can be done by slowly dialyzing again progressively weaker Urea solutions until you eventually get rid of the urea. The process has to be done slowly (stepwise) to avoid precipitation of the denatured proteins, until they have had a chance to re-fold.
The alternative is to use a non-detergent sulfobetaine in place of the Triton surfactant, typically 0.5 to 1 M. All surfactants cause the proteins to denature. So, again you will have to refold to get back to nature stuctures.
If these protein aggregates are truly an aggregate produced by the hydrophobic effect where internal amino acids participate, then only denaturing agents can be used to dissolve the protein while "denaturing it". The protein in the aggregate is already denatured.
Thanks for your suggestion Adam B Shapiro. I am planning to treat the cells with the Triton insoluble protein to see their effects on the healthy cells. For that I would like to keep the protein non-denatured form and so that we can claim that effect is true and physiologically relevant. Your suggestions are very welcome.
Thanks Luke V Schneider for your suggestion. I will try the refolding procedure but I wonder refolding will restore all the native characteristics of the protein which was before treated with Urea. Please kindly give me your thoughts.
Thanks Omar Gonzalez-Ortega but they may be misfolded and aggregate by the hydrophobic effect and I believe it is still misfolded protein aggregates and not denatured so that its need some special agent to solubilize the precipitates and then became denatured. I don't understand that "The protein in the aggregate already denatured" Please could you explain little bit more and I am bit new to this topic.
When you add any surfactant to a protein solution, the surfactant's tail binds to the hydrophobic regions of the protein and the pendant head group of the surfactant makes that more soluble in an aqueous solution. This alters the energetics of protein folding and disrupts the tertiary structure. Those proteins that precipitate in a surfactant solution are likely inverse structures where the surfactant head groups bind to hydrophillic regions of the protein and the tails form a micelle, causing precipitation. Again, the addition of the surfactant alters the energetics of protein folding, disrupting the tertiary structure. If you use any surfactant, you will alter the protein structure. Even NDSBs alter protein structure and activity. The advantage of NDSBs is that they don't bind as tenaciously to the protein as surfactants, therefore, are easier to remove by dialysis. I've done a lot of work with NDSBs, expensive, but much easier to recover proteins from them.
Try to use MPER, TPER or BPER reagents, otherwise nanodisc solutions may help. Belt like structure keeps membrane proteins native by providing lipid bilayer like environment...It may be a way assuming as unnecessarily aggresive, but could be helpfull if you should definitely omit using surfactants and must be find a solution other way around...
TD buffers also work to stabilize membrane proteins and extract them without surfactants when using a Barocycler.
Article Method for Recovery and Immunoaffinity Enrichment of Membran...
Samidurai Manikandan if you have a protein aggregate and not an agglomerate, you cannot dissolve the protein by gentle stirring with an aqueous buffer. The aggregate was formed when the buried hydrophobic amino acids from a protein somehow became exposed and protein molecules started to bind due to the hydrophobic effect, in this sense, this precipitate is already denatured protein. An agglomerate, on the other hand, is a precipitate that can be re-dissolved in a aqueous buffer with mild energy entrance. The protein molecules were bound weakly while preserving the tertiary structure. An aggregate can then only be dissolved by introducing chaotropic agents or surfactants. As I said before, the protein is already denatured, and you denature it even further to get it dissolved.
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