Video shots of the major and the most critical assay steps of a PREDEASYTM ATPase
Recommended equipment: | |
| 1. Water bath / heating block / thermoshaker (BIOSAN PST-60HL or equivalent), 37° C (Do not use CO2 incubator because of insufficient heat transfer!) | Video |
| 2. Automatic pipettes, multichannel-pipette with corresponding tips (Eppendorf Research, Multipette Plus or equivalent, Eppendorf pipette tips or equivalent quality tips). Please note that the membrane suspension and solutions are provided in excess quantity, still it might not be sufficient for electronic pipettes. | Video |
| 3. 96 well microtiter plates (COSTAR 3585 or equivalent) | Video |
Handling the membrane preparation | |
| 4. Homogenize the membrane stock with gentle pipetting. Gentle vortexing of diluted membrane suspension. (Step 5) | Video |
Incubation | |
| 5. Pre-incubate your plate and MgATP solution at 37°C for 10 minutes. (If the bottom of the wells do not touch directly the heated surface of your incubator, you may need a longer incubation time.) (Step 13) | Video |
Start the reaction | |
| 6. Start reaction by adding 10 µl MgATP solution to each well except of the wells of phosphate calibration. (Use a repeater pipette to minimize time of pipetting.) (Step 14) | Video |
Stop the reaction | |
| 7. Stop the ATPase reaction by adding Developer solution to the wells at room temperature. (Use 8-channel pipette or repeater pipette to minimize time of pipetting.) (Step 16) After two minutes add Blocker solution to the wells at room temperature. (Step 17) | Video |