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1. how to calculate the OD and results? 2. the source of "1x50ul Nuclear Extract(4mg/ml)". Is this HeLa cell extract? 3. Is it possible to purchase Nuclear Extract component of this kit?
Asked on Aug 16 2011
Thank you very much for your inquiry. 1.) I can confirm that the HAT activity can be expressed as the relative O.D. value per µg or nmol/min/µg sample. Please have a look at the following example calculation: A. Relative OD/ug: One obtains two readings at two time points in a linear region (from measuring OD kinetically) Example: suppose background (BG) and Sample (S) gave readings of 0.1 and 0.25 at T = 3 min and 0.12 and 0.6 at T = 30 min and sample was at 50 ug in 40 ul water. Then OD2 = 0.48 and OD1 = 0.15 (after subtracting background) so Rel Activity = (0.48-0.15)/(27*50) = 0.33 per min per ug. (27 is the time interval 30 min - 3 min) B. For nmol/min/ug: Need to first determine pathlength-which depends on the 96-well plate used and any meniscus from the buffers so: 1. First take any solution in the 1X HAT assay buffer (just dilute 1:1 with water) and adjust to read 1.0 OD in a 1 cm cuvette at a given wavelength (let’s say it was Coomassie blue and you measured at 595 nm); then take exactly 108 ul and place in a well of the 96-well plate you are using and measure the absorbance at 595 nm (and let’s suppose you get 0.25); so at a path length of 1 cm you got OD =1, at the well depth pathlength you got 0.25; so the pathlength is 0.25 cm 2. Now you know the pathlength in this buffer (which accounts for a meniscus effect from any detergent, etc. in the buffer). So Measure as in (a) above and let’s assume you get the same values as in (a) above: Then the diff in OD = 0.48-0.15 = 0.33; the time interval was 27 min; the amount of protein was 50 ug; the total volume was 108 ul and the datasheet tells you the extinction coefficient is 37000 M-1 cm-1. So: amount of product = (108 ul) x ((0.33)/(37000 x 0.25)) x (1L/1000000 ul) x (1000000000 nmol/mol)= 3.853 nmol and this was measured over 27 min and using 50 ug so = 3.853 nmol/(27 min x 50 ug) = 0.00285 nmol/min/ug. OR to convert to a common U (umol/min) and per mg would get the same value since 1000 nmol/umol and 1000 ug/mg cancel each other out so = 0.00285 U/mg 2.) I can confirm that the source of the NE buffer is Jurkat cells. 3.) I am sorry to confirm that we do not offer a Jurkat cell lysate suitable for this purpose at the moment. But I would like to reassure you that you will not need it for this kit, because the NE buffer acts as a positive control.
Answered on Aug 16 2011