This article discusses the preparation of organoid protein lysates from cultured organoids. The process involves using proteases and lysis buffers to extract proteins while preventing degradation.
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.
To collect organoids, repeatedly blast the matrix gel by pipetting 1 milliliter of pre-warmed dispase-containing media directly onto the matrix gel ring until the entire ring is dislodged. Transfer the dislodged matrix gel organoid mixture to a 1.5-milliliter microcentrifuge tube. Following complete digestion, as described in the text protocol, add phosphate-buffered saline to the organoid pellet and resuspend by gently flicking.
Pellet the organoids by centrifugation at 800 x g for 5 minutes at room temperature and remove the supernatant using a micropipette. Resuspend the organoid pellets in 100 microliters of protein lysis buffer per 10 microliters of packed cell volume. Flick to resuspend. Now, sonicate the organoids by submerging tubes in wet ice and gently applying the tip of the sonic dismembrator to the outside of the microcentrifuge tube. Sonicate for 40 seconds at 20 kHz before proceeding to western blotting with established protocols.
繁體中文
