Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides
筆記録
Begin with a prostate tumor tissue sample. Resuspend it in a chilled lysis buffer to initiate cell lysis. Use a homogenizer to break down the tissue structures further and release their intracellular components. Sonicate the sample on ice to ensure complete cell lysis and shear the DNA to prevent interference during peptide processing. Heat the lysate to disrupt the proteins' tertiary structure and unfold.
The reducing agents in the buffer cleave the disulfide bonds between cysteine residues. Subsequently, the alkylating agents in the buffer alkylate free thiol groups of reduced cysteines to prevent disulfide bond reformation. The additional denaturing agents in the buffer facilitate controlled denaturation and the further unfolding of proteins.
Centrifuge to pelletize the tissue debris and any contaminants. Collect the supernatant containing the protein mixture in a fresh tube. Add lysyl endopeptidase enzyme to cleave the peptide bonds in proteins at the C terminus of lysine residues, forming large peptides. Further, treat with the enzyme trypsin to cleave the peptide bonds at the C terminus of lysine and arginine residues and generate smaller peptides. Transfer the mixture to a centrifugal filter unit.
Centrifuge to allow the digested peptides to permeate through the pores of the filter membrane and collect as flow-through. Any larger fragments remain as the retentate.
