I am performing this assay with kinetic reading. The problem is that my initial blank fluorescence values are higher than those for my cell lysates. However, increase in fluorescence occurs only in my samples, not in blank.
The baseline activity matters little. The important thing in the delta in the accumulation of processed substrate in time.
For example, (Abs time 2 - Abs time 1) / time (minutes). Make many of these measurements every 5, 10 or 30 min. Convert Abs units into nmoles of DEVD substrate/mg protein/min. This is the enzyme activity.
In this particular case you will have to check the concentration/constituents of each component, for which the reading is taken. Also it may be the case that the cell lysate are too concentrated or opaque, that the clear reading is not possible. Is it the exact case...if not then let me know.
Dear Dianelena!
You look also following answer:
https://www.researchgate.net/post/Which_is_the_best_protocol_for_caspase-3_activity_detection_in_vitro
Patrik Erlmann added an answer
December 3, 2014
Ok, I might be wrong but I think that Ac-DMQD-AMC is not cell permeable... So that is what you need to do:
Treat your cells (6-well plate or 10cm dish) at the time point of interest put the cells on ice, lyse 10 min with a conventional lysis buffer (eg. 50mM Tris ph7.5, 150mM NaCl 0.5% Triton X100) but no!! protease inhibitors. Spin down unbroken cells and debris 10min at 10.000 rpm.
Incubate the cytosol fraction with Ac-DMQD-AMC. This is best done in a black 96 well plate... incubate from 10 to 30 min at RT and measure...
I would also include a positive control, e.g. Staurosporin works always.
Timings and amounts must be tested with your sample...
Oscar L. Sierra Thanks a lot! I´ve already done that measumerents and calculi, however, it is odd for me that blanks have high fluorescence values, since fluorescence is conferred by processed substrate.
Alexandr Chernov thank you for the information, I´ll check it carefully.
Dr. Satyam Kumar Agrawal thank you for your attention! My cell lysates are not concentrated, I think I may check each component, as you recommend, for finding where the unspecific signal comes from.
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