566328 HDAC Activity Assay Kit

566328
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      Overview

      Replacement Information

      Key Specifications Table

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      566328-1KIT
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          Plastic ampoule 1 kit
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          Description
          OverviewThe HDAC Activity Assay Kit is designed to measure HDAC activity in cell or nuclear extracts, immunoprecipitates or purified enzymes.
          Catalogue Number566328
          Brand Family Calbiochem®
          Application Data
          1Refers to dilution of Trichostatin A in HDAC Assay Buffer, which will be 5X the final concentration. Examples: 1) As a measure of non-HDAC background, 5 µM would produce final 1 µM concentration and essentially complete HDAC inhibition; 2) As a model inhibitor "hit", 25 nM would produce final 5 nM and ~50% inhibition. 2 Refers to dilution of potential inhibitor in Assay Buffer, which will be 5x its final concentration.

          1The appropriate dilution of the Deacetylated Standard may be determined from the standard curve and should be the concentration producing a fluorescent signal equal to that produced by control (no inhibitor) samples in the HDAC assay. The dilution in HDAC Assay Buffer is prepared at 1.25X this concentration to compensate for the 4/5 dilution due to addition of 10 µl of Assay Buffer or inhibitor. 2 Refers to dilution of Trichostatin A in HDAC Assay Buffer, which will be 5X its final concentration in the 50 µl volume, prior to addition of HDAC Developer WS. Example: As a model inhibitor that does not interfere with the HDAC Developer, 25 nM Trichostatin A would produce a final 5 nM concentration. 3Refers to dilution of potential inhibitor in HDAC Assay Buffer, which will be 5X its final concentration in the 50 µl volume, prior to addition of HDAC Developer WS.


          50 µl of each diluted Deacetylated Standard was mixed with 50 µl HDAC Developer and incubated for 10 min at 25°C. Fluorescence was then measured using the clear 1/2 Volume Plate with a fluorimeter (PerSeptive Biosystems, Ex. 360 nm, Em. 460 nm, gain=85)
          Materials Required but Not Delivered Fluorimeter for measuring fluorescence in a 96-well plate at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm.
          Pipetman or multi-channel pipetman capable of pipetting 2-100 µl accurately
          Ice bucket to keep reagents cold until use.
          Plate warmer or other temperature control device (optional)
          References
          ReferencesGurvich, N., et al. 2004. Cancer Res. 64, 1079.
          Kapustin, G.V., et al. 2003. Org. Lett. 5, 3053.
          Bitterman, K.J., et al. 2002. J. Biol. Chem. 277, 45099.
          Grozinger, C.M., et al. 2001. J. Biol. Chem. 276, 38837.
          Product Information
          Form96 Tests
          Format96-well plate
          Applications
          Biological Information
          Sample Typecell extracts, immunoprecipitates, or purified enzymes
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 36/38

          Irritating to eyes and skin.
          S PhraseS: 26-36-45

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          Product Usage Statements
          Intended useThe Calbiochem® HDAC Activity assay is a complete assay system designed to measure histone deacetylase (HDAC) activity in cell or nuclear extracts, immunoprecipitates, or purified enzymes. It has been used successfully with preparations of all class I HDACs-HDAC1, HDAC2, HDAC3 and HDAC8 - with class II HDACs 4-7, 9 and 10 and with yeast Sir2 and its human homolog, SIRT1.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage ≤ -70°C
          Storage ConditionsUpon arrival store the HeLa Cell Nuclear Extract, HDAC Substrate, HDAC Developer Concentrate, Trichostatin A (HDAC Inhibitor), Deacetylated Standard, and HDAC Assay Buffer at -70°C. Store both of the 1/2 Volume Microplates at room temperature.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Specifications

          Documentation

          HDAC Activity Assay Kit Certificates of Analysis

          TitleLot Number
          566328

          References

          Reference overview
          Gurvich, N., et al. 2004. Cancer Res. 64, 1079.
          Kapustin, G.V., et al. 2003. Org. Lett. 5, 3053.
          Bitterman, K.J., et al. 2002. J. Biol. Chem. 277, 45099.
          Grozinger, C.M., et al. 2001. J. Biol. Chem. 276, 38837.
          User Protocol

          Revision25-July-2012 JSW
          Form96 Tests
          Format96-well plate
          StorageUpon arrival store the HeLa Cell Nuclear Extract, HDAC Substrate, HDAC Developer Concentrate, Trichostatin A (HDAC Inhibitor), Deacetylated Standard, and HDAC Assay Buffer at -70°C. Store both of the 1/2 Volume Microplates at room temperature.
          Intended useThe Calbiochem® HDAC Activity assay is a complete assay system designed to measure histone deacetylase (HDAC) activity in cell or nuclear extracts, immunoprecipitates, or purified enzymes. It has been used successfully with preparations of all class I HDACs-HDAC1, HDAC2, HDAC3 and HDAC8 - with class II HDACs 4-7, 9 and 10 and with yeast Sir2 and its human homolog, SIRT1.
          BackgroundHistones form the protein core of nucleosomes, the DNA/protein complexes that are the subunits of eukaryotic chromatin. The histones' N-terminal "tails" are subject to a variety of post-translational modifications, including phosphorylation, methylation, ubiquitination, ADP-ribosylation, and acetylation. These modifications are believed to constitute a 'histone code' that, when translated, has profound regulatory functions in gene transcription. The most well-studied modification, acetylation of the ε-amino groups of specific histone lysine residues, is catalyzed by histone acetyltransferases (HATs). Histone deacetylases (HDACs) are responsible for hydrolytic removal of these acetyl groups. Histone hyperacetylation correlates to an open, decondensed chromatin structure and gene activation, while hypoacetylation correlates to chromatin condensation and transcriptional repression. Consistent with this, HATs have been shown to associate with several transcriptional activators and some transcriptional activators have been found to have intrinsic HAT activity. Conversely, HDACs are found to associate with transcriptional repression complexes such as NuRD or those including Sin3. Thus far, eleven human HDACs have been identified, all trichostatin A-sensitive and all homologs of either RPD3 (Class I HDACs) or HDA1 (Class II HDACs), yeast histone deacetylases. Interestingly, Sir2, the yeast mother cell longevity factor, and its mouse homolog, mSir2α, recently have been shown to be trichostatin A-insensitive, NAD+-dependent histone deacetylases. Human, archaeal, and eubacterial Sir2 homologs also display NAD+-dependent histone deacetylase activity. These enzymes apparently function via a unique mechanism, which consumes NAD+ and couples lysine deacetylation to formation of nicotinamide and O-acetyl-ADPribose. The Sir2 family (sirtuins) thus constitutes a third class of HDACs, but its members have yet to be included in the HDAC (Class I/Class II) numbering scheme. Histone deacetylase inhibitors have shown promise as antitumor agents, which and has stimulated interest in the screening of compounds for HDAC inhibition. Unfortunately, the standard techniques for HDAC assay are cumbersome. Use of [3H]acetyl-histone or [3H]acetyl-histone peptides as substrates involves an acid/ethyl acetate extraction step prior to scintillation counting. Unlabeled, acetylated histone peptides have been used as substrates, but reactions then require resolution by HPLC17.
          Principles of the assayThe Calbiochem® HDAC Activity Assay is supplied in a convenient 96-well format, with all reagents necessary for fluorescent HDAC activity measurements and calibration of the assay. In addition, a HeLa nuclear extract, rich in HDAC activity, is included with the kit. The extract is useful as either a positive control or as the source of HDAC activity for inhibitor/drug screening. Also included is the potent HDAC inhibitor, Trichostatin A, which may be used as model inhibitor.

          The assay is based on a unique HDAC Substrate and HDAC Developer combination, which is highly sensitive alternative to radiolabeled, acetylated histones or peptide/HPLC methods that are typically used to measure HDAC activity. The HDAC Substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (HeLa nuclear or other extract, purified enzyme, bead-bound immunocomplex, etc.). Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment with the HDAC Developer produces a fluorophore. The fluorescence is measured at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm.
          Materials provided• HeLa Cell Nuclear Extract (Kit Component No. KP31841-100UL): 1 vial, 100 µl, 6-9 mg protein/ml in 0.1 M KCl, 20 mM HEPES/NaOH, pH 7.9, 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF
          • HDAC Substrate (Kit Component No. KP31842-50UL): 1 vial, 50 µl, supplied as 50 mM in DMSO
          • HDAC Developer Concentrate (20X) (Kit Component No. KP31843-300UL): 1 vial, 300 µl, supplied as 20X
          • Trichostatin A (HDAC Inhibitor) (Kit Component No. KP31844-100UL): 1 vial, 100 µl, supplied as 200 µM in DMSO
          • Deacetylated Standard (Kit Component No. KP31845-30UL): 1 vial, 30 µl, supplied as 10 mM in DMSO
          • HDAC Assay Buffer (Kit Component No. KP31846-20ML): 1 bottle, 20 ml, 50 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂
          • 1/2 Volume Microplate (Kit Component No. KP31847-EA): 1 plate
          • 1/2 Volume White Microplate (Kit Component No. KP31848-EA): 1 plate
          Materials Required but not provided Fluorimeter for measuring fluorescence in a 96-well plate at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm.
          Pipetman or multi-channel pipetman capable of pipetting 2-100 µl accurately
          Ice bucket to keep reagents cold until use.
          Plate warmer or other temperature control device (optional)
          Precautions and recommendations The HeLa Nuclear Extract must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused extract should be refrozen quickly, by placing at -70°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot the extract into separate tubes and store at -70°C. The HDAC Activity Assay Substrate when diluted in Assay Buffer, may precipitate after freezing and thawing. It is best, therefore, to dilute only the amount needed to perform the assays of that day.
          The assay is performed in two stages. The first stage, during which the HDAC(s) acts on the HDAC Substrate, is done in a total volume of 50 µl. The second stage, which is initiated by the addition of 50 µl of HDAC Developer, stops HDAC activity and produces the fluorescent signal.
          Two types of 1/2-volume, 96-well plates are provided with the kit. The signal obtained with the opaque, white plate can be ~5-fold greater than that obtained with the clear plate. As long as the fluorimeter to be used is configured so that excitation and emission detection occur from above the well, the white plate should significantly increase assay sensitivity.
          Should it be necessary, for convenience in adding or mixing reagents, there is some flexibility for change in the reaction volumes. The wells of the microplates provided can readily accommodate 150 µl. If planning a change to the volume of the HDAC Developer, it should be noted that it is important to keep two factors constant: 1) the 1 µM concentration of Trichostatin A in the final mix; 2) 2.5 µl/well amount of HDAC Developer Concentrate.
          Experimental samples should be compared to a "time zero" (sample for which HDAC Developer is added immediately after mixing of the HDAC with HDAC Substrate) and/or a negative control (no enzyme).
          When 0.5 µl/well of the HeLa Cell Nuclear Extract is used as the source of HDAC activity, reaction progress curves, for a broad range of substrate concentrations (10-2000 µM HDAC Substrate), remain linear for at least 30 min (25°C). This will not necessarily be true if a different source of HDAC activity, a different amount of extract, or a different assay temperature is used. A time course experiment will aid in the selection of an incubation time, which yields a signal that is both sufficiently large and proportional to enzyme rate.
          The apparent Km of the HDAC activity in the HeLa Cell Nuclear Extract for the HDAC Substrate is ~50 µM. Use of a substrate concentration at or below the Km will help avoid substrate competition effects, which could mask the effectiveness of a potential inhibitor. If a different source of HDAC activity is to be used, a rate vs. concentration experiment should be performed before selecting a substrate concentration for the screening experiments.
          It is conceivable that some compounds being screened for inhibition of HDACs may interfere with the action of the HDAC Developer. It is therefore important to confirm that apparent HDAC inhibitor "hits" are in fact acting only via HDAC inhibition. One approach to this involves retesting the candidate inhibitor in a reaction with the Deacetylated Standard plus the HDAC Developer. A detailed retesting procedure is described below, "Uses Of The HDAC Activity Assay Deacetylated Standard". In some cases, it may be possible to avoid this retesting by means of measurements taken during the fluorescence development phase of the initial HDAC assay.
          Note that sirtuins (Sir2 and Sir2-like NAD+-dependent HDACs) are insensitive to Trichostatin A. Therefore, HDAC Developer prepared as described below with added Trichostatin A (HDAC Inhibitor), will not completely block further deacetylation by these enzymes. If the kit is to be used to assay a sirtuin, we recommend either reading the fluorescence at a consistent time shortly after addition of HDAC Developer (e.g. 2-5 min.) or adding a sirtuin inhibitor to the HDAC Developer. We recommend using the SIRT1 Activity Assay Kit (Cat. No. 569737) and the SIRT2 Activity Assay Kit (Cat. No. 566329) for measuring SIRT activity.
          Reagent preparationNote: Defrost all kit components and keep these, and all dilutions described below, on ice until use. All undiluted kit components are stable for several hours on ice. • Diluted HeLa Cell Nuclear Extract: Prepare a sufficient amount of HeLa Cell Nuclear Extract or other HDAC source diluted in HDAC Assay Buffer for the assays to be performed (# of wells x 15 µl). A 30-fold dilution of the HeLa Cell Nuclear Extract means that 15 µl contains 0.5 µl of the undiluted Extract, an appropriate amount to use per well (Table 1). • Trichostatin A (HDAC Inhibitor) WS: Prepare dilution(s) of Trichostatin A (HDAC Inhibitor) and/or Test Inhibitors in HDAC Assay Buffer. Since 10 µl will be used per well (Table 1), and since the final volume of the HDAC reaction is 50 µl, these inhibitor dilutions should be 5X their final concentration. • HDAC Substrate WS: Prepare dilution(s) of the HDAC Substrate in HDAC Assay Buffer that will be 2X the desired final concentration(s). For inhibitor screening, substrate concentrations at or below the Km are recommended. 25 µl will be used per well (Table 1). Initial dilutions of 25X or greater in HDAC Assay Buffer (2.0 mM or less) yield stable solutions (see NOTE on freezing and thawing below). Rapid mixing and dilution into room temperature buffer will help prevent precipitation at high substrate concentration. NOTE: Freezing/thawing of HDAC Substrate solutions in HDAC Assay Buffer may cause precipitation of the HDAC Substrate. Dilute only the amount necessary for one day's experiment. • HDAC Developer WS: Shortly before use (<30 min.), prepare sufficient HDAC Developer for the number of assays to be performed (50 µl/well). First, dilute the HDAC Developer Concentrate (20X) 1:20 (e.g. 50 µl plus 950 µl Assay Buffer) in cold HDAC Assay Buffer. Second, dilute the Trichostatin A (HDAC Inhibitor) 1:1000 in the HDAC Developer WS (e.g. 10 µl in 1 ml; final Trichostatin A (HDAC Inhibitor) concentration in the HDAC Developer = 2 µM; final concentration after addition to HDAC/Substrate reaction = 1 µM). Addition of Trichostatin A to the HDAC Developer insures that HDAC activity stops when the HDAC Developer is added. Keep Developer on ice until use.
          Detailed protocolStandard Curve Fluorimeter Preperation
          1. The exact concentration range of the Deacetylated Standard that will be useful for preparing a standard curve will vary depending on the fluorimeter model, the gain setting and the exact excitation and emission wavelengths used. We recommend diluting some of the Deacetylated Standard to a relatively low concentration with HDAC Assay Buffer (1-5 µM). The fluorescence signal should then be determined, as described below, after mixing 50 µl of the diluted Deacetylated Standard with 50 µl HDAC Developer. The estimate of AFU (arbitrary fluorescence units)/µM obtained with this measurement, together with the observed range of values obtained in the enzyme assays can then be used to plan an appropriate series of dilutions for a standard curve. Provided the same wavelength and gain settings are used each time, there should be no need to prepare a standard curve more than once.
          2. After ascertaining an appropriate concentration range, prepare, in HDAC Assay Buffer, a series of Deacetylated Standard dilutions that span this range. Pipet 50 µl of each of these dilutions, and 50 µl HDAC Assay Buffer as a 'zero', to a set of wells on the plate.
          3. Prepare, as described in "Reagent Preparation", sufficient HDAC Developer WS for the standard wells (50 µl/well).
          4. Mix 50 µl of the HDAC Developer WS with 50 µl of each standard in designated wells and incubate 5-10 min at room temperature (25°C).
          5. Read samples in a fluorimeter at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm.
          6. Plot fluorescence signal (y-axis) versus concentration of the Deacetylated Standard (x-axis). Determine slope as AFU /µM.

          HDAC Activity Assay
          1. Add HDAC Assay Buffer, Trichostatin A or test inhibitor to appropriate wells of the 96-well plate. Table 1 lists examples of various assay types and the additions required for each.
          2. Add Diluted HeLa Cell Nuclear Extract or other HDAC containing sample to designated wells except those that are to be "No Enzyme Controls."
          3. Allow HDAC Substrate and the samples to equilibrate to assay temperature (e.g. 25 or 37°C).
          4. Initiate 25 µl HDAC reactions by adding HDAC Substrate WS to each well, mix thoroughly.

          5. Allow HDAC reactions to proceed for desired length of time and then stop them by adding 50 µl HDAC Developer WS. Incubate the plate at room temperature (25°C) for 10-15 min. The fluorescent signal is stable for at least 30 min beyond this time.
          6. Read samples in a fluorimeter at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm.

          Testing of Potential HDAC Inhibitors for Interference with the HDAC Developer or the Fluorescence Signal:
          1. The HDAC Activity Assay Developer is formulated so that, under normal circumstances, the reaction goes to completion in less than 1 min at 25°C. That, together with the recommended 10-15 min reaction time, should help insure that in most cases, even when some retardation of the development reaction occurs, the signal will fully develop prior to the reading of the plate.
          2. Absolute certainty regarding interference with the HDAC Developer can only be obtained through an assay in which the compound in question is tested for its effect on the reaction of Deacetylated Standard with the HDAC Developer. Using a standard curve such as that described above, determine the concentration of Deacetylated Standard that will yield a signal similar to that produced after development of a control (no inhibitor) HDAC reaction. Mix 40 µl of the diluted Deacetylated Standard with 10 µl inhibitor or 10 µl HDAC Assay Buffer. Initiate development by adding 50 µl of HDAC Developer WS to each well. Follow fluorescence development by reading at 1 or 2 min intervals for 30 min. If a test inhibitor sample reaches its final fluorescence more slowly than the control or if the final value is significantly below that of the control, then there is interference with the HDAC Developer reaction.
          Standard curve
          Example dataThe HDAC Activity Assay has been used to investigate the kinetics of HDAC Substrate deacetylation by the HeLa Cell Nuclear Extract. This activity is nearly totally sensitive to the HDAC inhibitor Trichostatin A. HDAC1, HDAC2 and HDAC3, immunoprecipitated from the HeLa Cell Nuclear Extract and bound to protein A agarose beads, all deacetylate the HDAC Substrate, as do recombinant human HDAC8 and recombinant human Sirtuin 1. The HDAC Substrate is cell-permeable. Its deacetylation and the intracellular accumulation of its deacetylated form are trichostatin-sensitive. Note: The folowing example data is provided on as a guide. Optimal experimental conditions (e.g., concentrations of substrate and inhibitors, assay volume, buffer composition, incubation temperature, etc.) must be determined by the individual end-user.
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