HAT Assay Kit
Fluorescent(version B2)
Catalog No. 56100

Copyright 2009 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
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treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respectivecompanies.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Reagent Preparation
Assay Buffer AM1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Acetyl-CoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Recombinant p300, catalytic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Histone H3 Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Histone H4 Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Anacardic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
DMSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Stop Solution (Fluor). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Developer (Fluor) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocols
Preparation of Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Appendix
Section A. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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Overview
Active Motif’s fluorescent HAT Assay Kit is an easy and sensitive assay to either determine theactivity of your own source of purified histone acetyltransferases (HATs) or to screen potential
inhibitors of HAT activity.
The HAT Assay Kit contains a purified recombinant active p300 catalytic domain protein, histone
H3 and H4 N-terminal substrate peptides, anacardic acid as a control inhibitor, and all the essential buffers for a full 96-well plate assay.
This fluorescent HAT Assay works in three easy steps: first, the HAT protein is incubated withacetyl-CoA and either a histone H3 or histone H4 substrate peptide. During this reaction, the HAT
enzyme catalyzes the enzymatic transfer of acetyl groups from the acetyl-CoA to the histone peptide thereby generating the acetylated peptide and CoA-SH. The reaction is stopped by adding
stop solution, and after adding the complete developing solution, the developer reacts with thefree sulfhydryl groups on the CoA-SH to give a fluorescent reading of acetyltransferase activity1.

productformatcatalog no.

HAT Assay Kit (Fluorescent)1 x 96 rxns

56100

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1Introduction
Histone Acetylation
The basic structural unit of chromatin is the nucleosome, which consists of 146 base pairs (bp) ofDNA wrapped around a histone octamer. The histone octamer consists of two copies each of thecore histone H2A-H2B dimers and H3-H4 dimers, and the N-terminal tails of the histones are freefrom the bound core of the nucleosome structure where they can be modified by protein interactions in the nucleus. Histones, especially the N-terminal tails, are highly conserved from yeast tohumans, and the modifications are commonly acetylation, phosphorylation and methylation. The
modifications that occur at specific amino acid residues on the histone tails are very important for
downstream signaling events and are of much interest in recent studies1.
Histone acetylation is a modification that effects the nucleosome structure and therefore the ability of transcription factors to access the DNA and regulate gene expression. Recent studies havefound that hyperacetylation of histones can be associated with relaxed chromatin structure andactive gene transcription, while hypoacetylated histones can lead to transcriptional repression
by condensing the structure of chromatin and restricting the access of transcription factors to theDNA2.
Histone acetylation is a reversible process. Two superfamilies of enzymes are involved in histone
acetylation: histone acetyltransferases (HAT) and histone deacetylases (HDAC), which catalyze
the addition and removal of acetyl groups on histones. Histone acetyltransferases (HAT) areenzymes that catalyze the acetylation of lysine residues in the N-terminal tails of histone proteins.
The first nuclear HATs discovered were the general control of nuclear-5 (GCN5), and its paralog
p300/CBP accessory factor (PCAF). Both are conserved in organisms from yeast to humans,and GCN5/PCAF HATs have served as paradigms for the biochemical analysis of HAT structure,mechanism and function3. In addition to histones, GCN5/PCAF HATs target non-histone protein
substrates, such as transcription factors (i.e., p53) and structural proteins (i.e. α-tubulin). Morethan 20 HATs have been identified and these have been classified into one of five families(GNAT1, MYST (MOZ, YBF2/Sas3, Sas2, Tip60), TAFII250, nuclear receptor co-activators, andp300/cAMP response element-binding protein (CBP)). To date, a small number of HAT inhibitors
have been reported. A few natural small molecules such as anacardic acid, garcinol and curcumin
have been described as potent p300 and PCAF inhibitors4.

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Kit Performance and Benefits
The HAT Assay Kit (Fluorescent) is for research use only. Not for use in diagnostic procedures.
Recombinant p300 catalytic domain protein, activep300 catalytic domain is a purified recombinant human protein expressed in insect cells. p300
catalytic domain is supplied at 0.1 mg/ml. This recombinant protein corresponds to amino acids
965-1810 from the GenBank sequence accession number U01877.
Anacardic Acid

Arbitrary Fluorescence Units (AFU)

Anacardic acid is supplied at 28.7 mM in DMSO. Anacardic acid is a small molecule compoundfrom cashew nut shell liquid. 15 µM anacardic acid has been shown to quench HAT activity morethan 90%. The IC50 value of anacardic acid for p300 using histones as substrates was found tobe 8.5 µM and the IC50 value of anacardic acid for p300 using H3 peptide as the substrate wasfound to be 500 nM4.
40000
35000

Histone H3Histone H430000
25000
20000
15000
10000
5000
0Background

Anacardic Acid

p300

Garcinolp300 Activity

Figure 1: Effect of inhibitors on p300 HAT activity.
Fluorescent HAT Assay Kit results using 50 ng p300 catalytic domain per well with 50 µM Histone H3 peptide,
50 µM Acetyl-CoA, 15 µM Anacardic Acid, 25 µM Garcinol and 30 minute incubation at room temperature. The
background fluorescence was subtracted from the data.

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3Kit Components and Storage
The HAT Assay Kit (Fluorescent) is for research use only. Not for use in diagnostic procedures.
Store components at the temperatures indicated below. All components are guaranteed stable
for six months when stored at the indicated temperature.
Reagent

Quantity

Storage / Stability

5X Assay Buffer AM1

1.8 ml

-20°C to room temp.

Acetyl-CoA

105 µl

-80°C

50 µl
(0.1 mg/ml)-80°C

Histone H3 Peptide(MW=2068 Da)

0.275 µmoles (568 µg)-20ºC

Histone H4 Peptide(MW=1905 Da)

0.275 µmoles (524 µg)-20°C

Stop Solution (Fluor)

5 mlroom temp.

Developer (Fluor), 120X

84 µl

-20°C

Developing Solution (Fluor)

10 ml-20°C to room temp.

96-well plate

1 plateroom temp.

Anacardic Acid

4 µl
(10 mg/ml)-80°C to -20°C

1room temp.

Recombinant p300 catalytic domain

Plate sealer
Additional materials required• β-mercaptoethanol or Coenzyme A• DMSO• multi-channel pipette• multi-channel pipette reservoir• Fluorescent plate reader capable of exciting at 360-390 nm and reading emission at460-490 nm

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4Reagent Preparation5X Assay Buffer AM1
Prior to the assay, dilute to 1X with sterile water. Allow solution to warm to room temperature
before use. This assay buffer is used in the assay and for diluting the Acetyl-CoA, Histone peptide
substrates, and inhibitors. Prepare the quantity required for your assay including what you needto dilute the components. The diluted 1X Assay Buffer AM1 is stable for 3 months.
Acetyl-CoA
Acetyl-CoA is provided as a 6 mM stock solution. Prior to the assay, dilute 1:12 with the 1X Assay
Buffer to a final concentration of 0.5 mM. 5 µl are used per assay well. Use freshly diluted AcetylCoA for each assay.
Recombinant p300 catalytic domain
Avoid multiple freeze-thaws of the protein. Please aliquot and store frozen at -80°C if only a portion of the enzyme is needed for your assays.
Histone H3 Peptide and Histone H4 Peptide
Peptides are supplied lyophilized. Rehydrate each peptide with 2.2 ml of the 1X Assay Buffer
to obtain a 125 µM stock solution (125 pmol/µl). The peptide is supplied in a tube with a 1.5 ml
volume capacity. Start by adding a portion of the 2.2 ml 1X Assay Buffer to transfer the peptide toa new tube to resuspend in a 2.2 ml final volume. After the peptide is in solution, store at -20°C.
Use 20 µl per assay well for a 50 µM final concentration in the reaction. This concentration may
be reduced or increased with the 1X Assay Buffer if another final concentration is preferable.
Anacardic Acid
Anacardic Acid is supplied at 28.7 mM in DMSO. Dilute in 1X Assay Buffer AM1 to make a 150 µM
solution. For example, dilute 1 µl of Anacardic Acid stock solution in 190 µl 1X Assay Buffer. For a15 µM Anacardic Acid treatment, add 5 µl of the 150 µM solution per assay well.
DMSO
Prepare a DMSO dilution equivalent to the Anacardic Acid dilution to serve as a backgroundcontrol within the assay. Add 1 µl of DMSO to 190 µl of 1X Assay Buffer. Use 5 µl of diluted DMSO
per assay well.
Stop Solution (Fluor) Ready to use as supplied.
Developer (Fluor)
Developer stock is 3 mM in DMSO. Prior to use, dilute the developer 1:120 with Developing Solution to obtain a final developing solution concentration of 25 µM. Protect the Developer and final
Developing Solution from light. The final diluted developing solution is stable for 6 hours at roomtemperature.
Note:

The assay will not work if the developer is diluted in assay buffer instead ofdeveloping solution.

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5Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Minimize the use of sample buffers containing DTT or β-mercaptoethanol for your HAT source.
Buffers containing these reducing agents or other sources of reactive thiol groups will result inhigh background.
This protocol has been optimized for use with recombinant proteins. If you wish to calculate
enzymatic activity, a standard curve must be prepared.
It is recommended to run test samples, controls, standards and background wells at least induplicate.

Preparation of Standard Curve (Optional)
If you want to calculated enzymatic activity, you will need to run a standard curve alongside yoursamples in the assay. A standard curve may be prepared with Coenzyme A or β-mercaptoethanol.

Arbitrary Fluorescence Units (AFU)

For example, to prepare a standard curve with β-mercaptoethanol, make dilutions in duplicate
ranging from 0 to 14 µM in 2 µM increments in a 50 µl volume to result in a 0 to 500 pmol range
for the X-axis (Figure 1). The volumes below are enough for duplicate wells.

25000

β-mercaptoethanol
Acetyl-CoA

20000
15000
10000
R2= 0.99
y = 26.49x + 1120.6

5000
00200

400

600

800pmol

Figure 1: Standard Curve Data.
Example standard curve data of both acetyl-CoA and β-mercaptoethanol diluted from 0 to 14 µM. pmol of
β-mercaptoethanol = µM of β-mercaptoethanol X 50 µl per wellRun standard curve wells by adding 50 µl of each dilute per well in duplicate and continue withstep 4 in the following protocol.
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6Assay Protocol
1.

Design your assay to test all samples in duplicate. Prepare the quantity required of 1X Assay
Buffer, diluted Acetyl-CoA and desired inhibitors. If using the Anacardic acid inhibitor provided in the kit, prepare a DMSO dilution for background comparison. Prepare the amountof final developing solution required and keep protected from light. The HAT enzyme andAcetyl-CoA should be kept on ice.

2.

Using any desired microcentrifuge tubes for samples including background, positive control
and inhibition wells, gently mix the following components together in the quantities indicated. 30 µl of each mix is needed per assay well. Keep the reactions at room temperature.
The volumes listed in the following table are enough for duplicate wells.
Reagent

Background
Wells

PositiveControl
Wells

Inhibitor
Wells

1X Assay Buffer

48.75 µl48.75 µl48.75 µl

0.5 mM Acetyl-CoA

12.5 µl12.5 µl12.5 µl

150 µM Anacardic AcidStandardCurve
Wells

12.5 µl

Diluted DMSO

12.5 µl12.5 µlp300 catalytic domain (0.1 mg/ml)

1.25 µl1.25 µl1.25 µl

30 µl

30 µl

30 µl

β-mercaptoethanol standard
Total volume per well3.

120 µl50 µl

Add 20 µl of Histone H3 or Histone H4 peptide to each assay well. Do not add peptide
substrate to background wells so that autoacetylation activity of the acetyltransferase isincluded in the background measurement. Mix solution by pipetting contents up-and-downwith multichannel pipettor. Cover plate with plate sealer and incubate for 10 to 30 minutesat room temperature.
Note:

If you are determining the activity of a new HAT protein, we recommend runninga time course to establish the correct incubation time for your protein.

4.

Add 50 µl of Stop Solution per well. To background wells, add 20 µl peptide substrate after
adding the stop solution.

5.

Add 100 µl final Developing Solution to each well and incubate 15 minutes in the dark atroom temperature.

6.

Read fluorescence with excitation at 360-390 nm and emission at 450 to 470 nm.

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7Data Analysis
Plot the standard curve data with fluorescence units on the Y-axis and pmol of β-mercaptoethanolon the X-axis.
Determine slope of the line through the linear part of the curve as arbitrary fluorescence units perpmol. Subtract the fluorescence of background from that of the assay samples and divide the difference by the incubation time (AFU/minute). Divide the AFU/minute by the slope of the standard
curve (AFU/pmol) to obtain the rate in pmol/minute.

References
1.
2.
3.
4.

Trievel, R. C., et al., (2000) Analytical Biochemistry 287: 319-328.
Wang, L., et al., (1997) Mol. and Cell. Biol. 17(1): 519-527.
Marmorstein, R. (2001) Cell. Mol. Life Sci. 58: 693-703.
Balasubramanyam K, et al., (2003) J. Biol. Chem. 278(21):19134-19140.

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Appendix
Section A. Troubleshooting Guide
Problem/question

Recommendation

Must the assay be incubated at room temperature?

The assay may be performed at 30˚C or room temperature without effecting results.

Is it possible to use diluted
Acetyl-CoA?

No. Fresh Acetyl-CoA should be prepared for each assay.

Is it necessary to run astandard curve?

No. The standard curve is only necessary if the specific activity needs to be calculated(pmol/min/µg).

Why was no HAT activity
detected?

The recombinant p300 supplied in this kit may lose activity after 3 freeze-thaws. Pleasealiquot at first use to store and reduce the number of freeze-thaws.
Check that the developer has been diluted into developing solution. Do not use Assay
Buffer AM1 to dilute!

Why is there high background?

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The background is due to the free sulfhydryl groups in the formulation buffer. Some background may be due to the protein’s auto-acetylation activity. Run controls with and without
acetyl-CoA to determine background and auto-acetylation levels.

9Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

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