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Protokoll
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A: Reagent Preparations and General Precaution: Reconstitute HAT Substrate I, substrate II, and NADH Generating Enzyme each with 550 μ l HAT Reconstitution Buffer. The substrate II will be become brown cloudy, and milky color. Pipette up and down several times to dissolve. The reagents are stable for two month at –80 o C after reconstitution. Nuclear Extract or purified protein samples can be tested using this kit. For nuclear extract preparation, please refer to the Nuclear/Cytosol Fractionation Kit (BioVision, K266-100) without using DTT, as DTT interferes with the assay. Samples containing DTT, Coenzyme A, and NADH should be avoided, as these compounds strongly interfere with the reactions. Use U-shape 96-well plates may increase signal up to 40% in comparison to the flat shape plates. B. HAT 1. Prepare test samples (50 μ g of nuclear extract or purified protein) in 40 μ l water (final volume) for each assay in a 96-well plate. For background reading, add 40 μ l water instead of sample. For positive control, add 10 μ l of the NE (Cell Nuclear Extract) and 30 μ l water. 2. Assay Mix preparation: Mix enough reagents for the number of assays performed. For each well, prepare a total 65 μ l Assay Mix containing: 50 μ l of 2X HAT Assay Buffer 5 μ l HAT Substrate I 5 ul HAT Substrate II (Mix before use) 5 μ l NADH Generating Enzyme 3. Mix the prepared Assay Mix, add 65 μ l of Assay Mix to each well, mix to start the reaction. 4. Incubate plates at 37 o C for 1 ~ 4 hours depending on the color development. Read sample in a plate reader at 440 nm. For kinetic studies, read O.D. 440nm at different times during incubation. Notes: 1) The yellow color develops slowly, but very steady and repeatable. 2) Background reading from buffer and reagents (without HAT) is significant, which should be subtracted from the readings of all samples. 3) HAT activity can be expressed as the relative O.D. value per μ g or nmol/min/ μ g sample. ε 440nm = 37000 M -1 cm -1 under the kit assay conditions. Advantages: The HAT Activity Colorimetric Assay (K332-100) provides an easy and very simple procedure to assay HAT activity (just adding reagents to sample preparations, incubate and read). Unlike the conventional radioisotope method, the assay continuously measures HAT activity and thus is suitable for kinetic studies. In addition, the assay is not interfered by the presence of histone deacetylases and therefore, crude nuclear extract can be used directly in the assay.
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