I wanted to see if people had experience and success with this kit without the use of a liquid dispenser.
Currently, I have had success with doing the direct enzyme + ATP and observing a good response curve. However, the inhibitor titration has given me zero success. I do not have a functional liquid dispenser (I can get access to one if needed), so I depend on manual pipetting.
As a result, the resulting luminescence output is absolutely non-sensical and has no observable trend in the numbers. Further, even my no enzyme duplicates have high values (not higher than those with enzyme, but still higher than they should be). A person from technical support has tried to help, but seems to be uncertain where my issues are coming about.
Sorry for the novel here, but I want to know if people have successfully used or done troubleshooting on this kit and, if so, how. I can show you my experimental details (materials and the like) and a sample output, if people would like. Just let me know.
Thank you!
I have used ADP-Glo successfully, using hand-held pipettors.
Are you using the ultra-pure ATP supplied with the kit? Have you done an ADP standard curve?
Hey Adam,
Yes, I have used only the materials from the kit aside from the DTT, which I have had to make, but it should be pure enough. (Admittedly, the ATP might be getting old, but these are the original aliquots I made when the kit first arrived.)
Having said that, the ATP is the same that the kit provided, but it certainly does not pardon it from fault. I made a standard curve initially, which worked, but have tried to use the SB10 from that curve and have not had any success. I could do another one and see if it is not the enzyme.
Any other thoughts given this information?
It might help me identify problems if you share some of your results.
Sorry for the delay. I obtained a new kit in the event that perhaps one of the components became contaminated during my attempts.
As such, you will find my most recent enzyme titration along with a calibration curve. From these data, I determined an "SB10" of 9.38 ng of my enzyme. The other file attached is the competition data I obtained using the same ATP stock I made from the generated calibration curve. I am uncertain how to interpret these data, but needless to say, there is no identifiable trend here. Is the ADP-Glo not quenching the reaction in the time I am allowing? Is it actually a problem of pipetting?
For reference (in this data attached), I have actually gone back and prepared my inhibitor titrations in smaller microcentrifuge tubes and done my titrations there. Further, it was suggested by a Promega tech representative to prepare slightly more kinase solution, put 8 µL of it into separate microcentrifuge tubes, aliquot the respective 4 µL of inhibitor titration into the tube containing the kinase solution, incubate for 10 minutes, and then aliquot 3 µL into the plate where they would otherwise be aliquoted in separately and at smaller volumes individually.
As you will see, this did not actually change my result at all. (Up until this point, I had been following the protocol as stated, pipetting the kinase solution into a preparative row and then aliquoting 2 µL in all of the assay rows aside from wells B23 and 24.)
Let me know what you think, however, it is clear to me that the the calibration and enzyme titration worked well. It is yet unclear to me why the inhibitor tests do not work. These are fresh Staurosporine stocks that were prepared, aliquoted into HPLC vials, sealed, Parafilm'd and then stuck in a -20 ºC freezer. Admittedly, the pipettes I am using, while calibrated, are seemingly the least capable at pipetting small volumes (Rainin) and I may resort to taking pipettors from my old lab that has well-calibrated Eppendorf Research Plus with Eppendorf low retention tips.
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