I am trying to figure out the best method to prepare vesicles of MDR1 (Pgp) for an ATPase assay and do not know the best method to use for preparing these vesicles. They have been growing in flasks with a monolayer of cells.
You can prepare a total membrane extract of the cells by sonication followed by ultracentrifugation, then resuspend the membrane pellet. You can also use that material as the starting point for purification of subcellular fractions by sucrose density gradient ultracentrifugation. For example, you can prepare plasma membrane vesicles. A method is given here:
https://www.jbc.org/article/S0021-9258(17)41923-5/pdf
Membranes contain multiple ATPases, so you will have to include some inhibitors such as ouabain and sodium orthovanadate, as described here:
Article P-Glycoprotein-Mediated Hoechst 33342 Transport Out of the L...
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes