Protein Lysate Extraction: A Technique to Lyse Organoids to Collect Cellular Proteins
Transcription
Cell lysate, or the fluid containing lysed cell contents, finds application in downstream processes like protein extractions and analyses. Studying these fractions reveals information about the characteristics and functions of cells.
To prepare organoid protein lysates, begin with a culture of organoids grown in a desired basement membrane matrix. Remove the spent culture media from the dish. Add warm media containing proteases over the matrix. Pipet repeatedly to dislodge the matrix from the dish.
Incubate the mixture. The proteolytic enzymes in the media degrade the matrix and release the organoids into the solution. Centrifuge the sample to separate organoids from the enzyme-containing supernatant.
Resuspend the organoid pellet in a suitable protein lysis buffer. Incubate the sample. The detergents in the buffer disrupt the organoids' cell membranes, releasing intracellular contents into the solution. Concurrently, the protein inhibitors in the buffer inactivate any released protease and phosphatase enzymes, preventing degradation of their substrate proteins.
Further, sonicate the mixture to disrupt the cells completely. The nuclear envelope ruptures and releases nuclear proteins into the solution. The sonic waves also shear the nucleic acids released, preventing them from contaminating the protein lysate.
