i've extracted protein from trizol samples using manufacturers method of collecting phenol-chloroform layer and washing in isopropanol, guanindine hydrochloride, ethanol then resuspending in 1% SDS. the pellet will not dissolve even after heating in SDS at 50deg celcius for 45mins. The pellet firs formed after initail washing in isopropanol .
Dear Sir. Concerning your issue about how do to dissolve a protein pellet extracted from trizol samples. Solvents used to solubilize the proteins in the pellets are two solvents that resuspended the largest portion of the dialyzed pellets were 8 M urea in Tris-HCl, pH 8.0, and aqueous 1%. Urea and SDS are frequently used for solubilizing proteins. Urea is a powerful protein denaturant; it breaks disulfide bonds and increases the solubility of some proteins, although the carbamylation of proteins has been demonstrated as a potential undesirable secondary effect. As a detergent, SDS aids in disrupting noncovalent bonds in the proteins, thereby denaturing them and causing the molecules to lose their native conformation. To facilitate protein solubilization in SDS, the solution can be boiled for short periods of time without protein degradation. While we tried all of these approaches, the best solubilization was achieved when the 8 M urea in Tris-HCl, pH 8.0, and aqueous 1% SDS were combined in a 1:1 ratio. I think the following below links may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721573/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721573/pdf/nihms663109.pdf
https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
Thanks
Isam Eldin Hussein Elgailani ·
thank you for your suggestion. I have limited access to fresh tissues as such I would like to salvage protein already isolated. My undissolved pellets are frozen in -20deg can this be used and heated 95deg in the solvent you recommended above? How long should it be heated for?
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