Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352)

You can use a background control sample, also use a nuclear extract.

I have had the chance to inquire to the lab about the use of a purified IP sample. Theywould suggest using the initial sample without any IP for the HAT assay. If the IP is just for concentrating the sample, they recommend use of spin columns, not IP.

Theoretically though, the IP samples should also work they do not have any experience using this method and though modifications to the protocol may have to be performed to optimize the reaction, these would have to be determine experimentally in the lab.

The lab has confirmed that this kit has not been validated for measuring Tubulin Acetyltransferase activity.  We have no information about whether it would be suitable for this purpose, but the kit was specifically designed for measuring Histone Acetyltransferase Activity. 

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.

DDT should be avoided, as this compound strongly interferes with the reactions in the kit. There are a few things I would like to suggest: - After acid extracting the histones in your current protocol, the sample could be dialysed overnight in 1 x TBS with 3,500 MWCO dialysis tubing. This would neutralize the sample. - You could use our nuclear fractionation protocol, but exclude DTT from the buffers. Here is a link to the protocol: https://www.abcam.com/index.html?pageconfig=resource&rid=11408 - Our cell fractionation kit, ab109719 Cell Fractionation Kit Standard, could be used: https://www.abcam.com/Cell-Fractionation-Kit-Standard-ab109719.html The nuclear extraction buffer in this kit contains SDS and DTT to generate a nuclear fraction for WB. Therefore this fraction is not compatible with a subsequent enzymatic analysis (though the other fractions are active). However, it may be possible to analyze the nuclear fraction without adding this buffer. Simply add water to the N fraction or treat with RIPA buffer : 1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM b-glycerophosphate 1 mM Na3VO4 1 μg/ml leupeptin

As long as the tissue samples were frozen appropriately, nuclear lysates generated from these tissues can be used for the HAT assay.

The feedback from the lab is that technically BME should be fine but we have not tested this specifically.

Thank you for your enquiry. I have obtained the following information to help answer your questions: 1) Each kit can test 50 samples. i.e. 100/2, minus the wells for background and the Hela nuclear extract used for a standard 2) If you use a spectrophotometer at 405 nm, whether you will be able to accurately read the results will depend on the bandwidth of the filter your reader has. If it has a 30 nm bandwidth that would cover 440 nm; if the band width is very small, may not be optimal. I can recommend to check this. 3) I can suggest that whole cell extracts will probably give more background and the results may not be as clear. As HAT is a nuclear protein, we would recommend to try nuclear extract.