Protein Lysate Extraction: A Technique to Lyse Organoids to Collect Cellular Proteins
Transcrição
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.
