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Hi there Please find attached data from the PDE12 assay (Abcam protein ab116283, recieved on the 20th Oct, 2011). Increase in delta fluorescence over 2h suggests that PDE12 at lower concentration (0.2ug to 0.4ug) is active however its not significantly active, compared to the positive lysate (which is from HepG2 cell line and is not true representative). I wonder if its possible to have an aliquot of the cell lysate from which your lab purified PDE12. I could then repeat the assay along with cell lysate from your lab and calculate PDE12 activity. Please let me have your thoughts and comments. Regards
Asked on Nov 01 2011
Thank you for your patience and for your feedback. I am very sorry to hear that the new protein does also not show enough activity for being used in an activity assay. In regards to use the whole cell extract, I am not very optimistic to be honest. I am not sure whether the lab is able to provide it, and even if so, it would be complicated and take time. More importantly however, I am not very hopeful that the lysate would have activity if the purified protein does not show activity. and I do not have much hope that the transfected cells would give a better result. Indeed, in the cell extract there are many additional factors which could inhibit the activity of the protein. I am sorry for not being able to be more optimistic on this. I would actually suggest to search for a PDE12 protein produced in a different system. I have asked my colleagues to check with their collaborators whether there might be another PDE12 protein available - maybe even a PDE12 protein tested for functional activity. I will let you know as soon as I have received the answer. Meanwhile, can you please also submit the Abreviews for the two proteins? We would greatly appreciate if you could put there also a summary of the test procedure in the additional notes. Thank you very much! Please let me know your thoughts and if you have any questions. I am looking forward to hear back from you.
Answered on Nov 01 2011