I would like to perform an ATPase assay on purified dynein, however I am totally clueless on what should be the concentration of the pure protein with which I perform the assay. Any suggestions or help regarding already published work explaining the same will be of great help!
Protein amount will vary with activity associated with the source. Make a dose response curve of ATPase activity with your protein, and find appropriate concentration from the linear part.
I would say it is important to know what ATPase assay you are going to perform. Typically you will need a low concentration of protein (10-100 nM) and you will perform the ATPase assay at various ATP concentrations 1 uM - 1 mM. The key aspect regarding concentration of the protein is what assay you are going to use because the sensitivity varies. For example, if your ATPase has a rate (kcat) of 10 s-1 then 10 nM enzyme will produce 100 nM ADP per second. Run the assay for 1 minute and you have 6 uM ADP produced, 12 uM for 2 min etc. But if the kcat is only 1 s-1 then after 1 min you will only generate 600 nM ADP etc etc. Therefore, the sensitivity of the assay (and length of time it runs for) is important to decide on a concentration.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays