Same answer as before! See attached. Why are you reposting the same question (not happy with the answer?)?

https://www.researchgate.net/post/Total_proteins_extraction

Dear Sir. Concerning your issue about protocol for total proteins extraction from human tissues. Maximal extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis. It also helps to maximize reproducibility and to minimize waste. Minimal degradation of the protein amino acid backbone or dephosphorylation is essential to preserve the analytical utility of the extract. Containment of the sample is important to minimize the risk of contamination to and from the sample. The proposed standard protocol for protein extraction and solubilization can result in 98% solubilization of brain tissue, corresponding to about 100 μg protein per mg tissue wet weight, by a frozen disintegration/SDS-based solubilization method: Tissue is crushed in the frozen state in a cryotube by shaking with a sterile steel ball. The crushing is followed by the extraction and solubilization in 2% SDS for 10 min, at 70°C, in a volume corresponding to ten times the tissue wet weight, with shaking. The containment in a cryotube helps to prevent contamination. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. The resulting protein extracts can be used for SDS PAGE, 2-D PAGE, Western blotting, ESI-MS, and ELISA. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification, and quantitation from cryopreserved clinical samples are desirable. I think the following below links may help you in your analysis:

https://www.gbiosciences.com/image/pdfs/protocol/786-225_protocol.pdf https://www.ncbi.nlm.nih.gov/pubmed/20949398

Thanks