HAT Activity Colorimetric Assay Kit
Contents
1. BioVision HAT Activity Colorimetric Assay Kit Catalog K332 100 100 assays Store at 80 C Introduction Histone acetyltransferases HATs have been implicated in a crucial role in various cellular functions such as gene transcription differentiation and proliferation BioVision s HAT Activity Colorimetric Assay Kit offers a convenient nonradioactive system for a rapid and sensitive detection of HAT activity in mammalian samples The kit utilizes active Nuclear Extract NE as a positive control and acetyl CoA as a cofactor Acetylation of peptide substrate by active HAT releases the free form of CoA which then serves as an essential coenzyme for producing NADH NADH can easily be detected spectrophotometrically upon reacting with a soluble tetrazolium dye The detection can be continuous and suitable for kinetic studies The kit provides a simple straightforward protocol for a complete assay Kit Contents Rev 06 11 K332 100 Component 100 assays Cap Color Part Number 2X HAT Buffer 7 5 ml Amber K332 100 1 HAT Substrate 1 vial Blue K332 100 2 HAT Substrate II 1 vial Purple K332 100 3 NADH Generating Enzyme 800 ul Green K332 100 4 Nuclear Extract NE 4 mg ml 50 ul Red K332 100 5 HAT Reconstitution Buffer 1 8 ml Clear K332 100 6 Reagent Preparation and General Precaution e Reconstitute HAT Substrate substrate II with 550 ul HAT Reconstitution Buffer The Substrate II will be become brown cl2. e Use fresh samples or store at correct temperatures until use ee in Samples Improperly thawed components Thaw all components completely and mix gently before use e Use of expired kit or improperly stored reagents e Always check the expiry date and store the components appropriately e Allowing the reagents to sit for extended times on ice e Always thaw and prepare fresh reaction mix before use e Incorrect incubation times or temperatures e Refer datasheet amp verify correct incubation times and temperatures e Incorrect volumes used e Use calibrated pipettes and aliquot correctly enia F ENER angang Use of partially thawed components Thaw and resuspend all components before preparing the reaction mix e Pipetting errors in the standard e Avoid pipetting small volumes e Pipetting errors in the reaction mix e Prepare a master reaction mix whenever possible e Air bubbles formed in well Pipette gently against the wall of the tubes e Standard stock is at an incorrect concentration e Always refer the dilutions in the data sheet e Calculation errors e Recheck calculations after referring the data sheet e Substituting reagents from older kits lots e Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength e Check the equipment and the filter setting Samples contain interfering substances e Troubleshoot if it interferes with the kit e Use of incompatible sample type e Refer data sheet to check if sample is compatible wi
3. oudy and milky color Pipette up and down several times to dissolve The reagents are stable for two months at 80 C after reconstitution e Nuclear Extract or purified protein samples can be tested using this kit For the nuclear extract preparation please refer to the Nuclear Cytosol Fractionation Kit BioVision K266 100 without using DTT as DTT interferes with the assay e Samples containing DTT Coenzyme A and NADH should be avoided as these compounds strongly interfere with the reactions e Using U shaped 96 well plates may increase signal up to 40 in comparison to flat bottom plates HAT Assay Protocol 1 Prepare test samples 50 ug of nuclear extract or purified protein in 40 ul water final volume for each assay in a 96 well plate For background reading add 40 ul water instead of sample For positive control add 10 ul of the NE Cell Nuclear Extract and 30 ul water 2 Assay Mix preparation Mix enough reagents for the number of assays performed For each well prepare a total 68 ul Assay Mix containing 50 pl 2X HAT Assay Buffer 5 ul HAT Substrate 5 ul HAT Substrate II Mix before use 8 ul NADH Generating Enzyme 3 Mix the prepared Assay Mix add 68 ul of Assay Mix to each well mix to start the reaction 4 Incubate plates at 37 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 BioVision Inco
4. rporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only Notes 1 The yellow color develops slowly but very steadily 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 ug or nmol min yg sample 4sonm 37000 Mcm 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 04 0 3 4 0254 O D 440 nm 017 2 h 8 10 12 E HeLa Nuclear Extract ul assay Fig 1 Analyses of HAT Activity in HeLa Nuclear Extract HeLa nuclear extract Cat 1641 1 in various amounts was incubated with HAT substrate Activity was analyzed in a micro plate reader at 440 nm according to the kit instructions RELATED PRODUCTS Cell Fractionation System Mitochondria Cytosol Fractionation Kit Nuclear Cytosol Fractionation Kit Cytosol Particulate Rapid Separation Kit FractionPREP Fractionation Sys
5. tem Cell Proliferation amp Senescence Assays Cell Damage amp Repair HDAC Fluorometric amp Colorimetric Assays amp Drug Discovery Kits DNA Damage Quantification Kit and more FOR RESEARCH USE ONLY Not to be used on humans Fax 408 493 1801 tech biovision com Tel 408 493 1800 www biovision com Page 1 of 2 BioVision Rev 06 11 GENERAL TROUBLESHOOTING GUIDE Problems Cause Solution Assay not working e Use of ice cold assay buffer e Assay buffer must be at room temperature e Omission of a step in the protocol e Refer and follow the data sheet precisely e Plate read at incorrect wavelength e Check the wavelength in the data sheet and the filter settings of the instrument Use of a different 96 well plate P Black plates clear bottoms Luminescence White plates Colorimeters Samples with erratic readings e Use of an incompatible sample type e Refer data sheet for details about incompatible samples Samples prepared in a different buffer e Use the assay buffer provided in the kit or refer data sheet for instructions Cell tissue samples were not completely homogenized Use Dounce homogenizer increase the number of strokes observe for lysis under microscope Samples used after multiple free thaw cycles Aliquot and freeze samples if needed to use multiple times e Presence of interfering substance in the sample Troubleshoot if needed e Use of old or inappropriately stored samples
6. th the kit or optimization is needed Sample readings above below the linear range Concentrate Dilute sample so as to be in the linear range Note The most probable list of causes is under each problem section Causes Solutions may overlap with other problems BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA www biovision com tech biovision com Page 2 of 2
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