Instructions for Use
For the measurement of activity/inhibition of H3K9 specific histone demethylases using cell/tissue extracts
This product is for research use only and is not intended for diagnostic use.
Version 1 Last Updated 5 September 2014

INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. SAMPLE PREPARATION
ASSAY PROCEDURE
11. ASSAY PROCEDURE
DATA ANALYSIS
12. ANALYSIS
RESOURCES
13. TROUBLESHOOTING
14. NOTES
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INTRODUCTION
Lysine histone methylation is one of the most robust epigenetic marks, and is essential for the regulation of multiple cellular processes. The methylation of H3-K9 seems to be of particular significance as it is associated with repression regions of the genome. H3K9 methylation was considered irreversible until identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics. So far, at least 2 classes of H3K9 specific histone demethylase – JMJD1 (JHDM2) and JMJD2 (JHDM3) – have been identified. The JMJD1 family (including JMJD1A, JMJD1B, and JMJD1C) can remove di- and mono-methylation from H3K9; while the JMJD2 family
(including JMJD2A, JMJD2B, JMJD2C, and JMJD2D) can remove trimethylation from H3-K9. Both, JMJD1 and JMJD2, catalyze the removal of methylation by using a hydroxylation reaction with a required iron and ketoglutarate as cofactors. Lastly, H3K9 specific demethylases are found to be involved in some pathological proc-esses such as cancer progression.
Inhibition of the enzymes may lead to re-methylation of H3K9 and activation of H3K9 enriched repression genes. Currently, there are few methods available for measuring activity/inhibition of H3K9 specific methylases using a variety of cells/tissues.
ab113458 uses a proprietary and unique procedure to measure activity/inhibition of H3K9 specific histone demethylases using cell/tissue extracts.
This kit has the following features:
 Fast procedure, which can be finished within 1.5 hours.
 Innovative fluorescent assay without the need for radioactivity, extraction, or chromatography.
 Measurement of HDM (H3K9 specific) activity and inhibition through quantifying by-product of the enzyme reaction, which makes the assay highly sensitive.
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INTRODUCTION
 Strip microplate format makes the assay flexible: manual or high throughput analysis.
 Simple, reliable, and consistent assay conditions.
The Histone Demethylase H3 (K9) Activity Quantification Assay Kit is designed for measuring total histone demethylase (H3K9 specific) activity/inhibition. In the assay with this kit, the unique methylated histone
H3K9 substrate is incubated with nuclear extracts or purified enzymes.
Formaldehyde, generated from the enzyme-substrate reaction, reacts with a detection reagent to form a fluorescent substance. The ratio or amount of the formaldehyde, which is proportional to HDM enzyme activity, can then be fluorometrically quantified.
Nuclear Extract
Incubate with substrate and assay buffer
Add detection reagent
Fluorescence measurement
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GENERAL INFORMATION
Please read these instructions carefully prior to beginning the assay.
All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance.
Store kit as given in the table upon receipt and away from light.
Observe the storage conditions for individual prepared components in sections 9 & 10. The components of the kit are stable for 6 months when stored properly.
For maximum recovery of the products, centrifuge the original vial prior to opening the cap.
Tightly cap Fluoro-Developer after each use.
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GENERAL INFORMATION
Item
HDM Assay Buffer
HDM Substrate
HDM Standard, 10 mM*
Fluoro Developer*
Fluoro Enhancer*
HDM Cofactor 1*
HDM Cofactor 2*
HDM Cofactor 3*
8-Well Assay Strip (with Frame)
2 mL
50 µL
25 µL
4 mL
4 mL
25 µL
25 µL
25 µL
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Quantity (48 tests)
Quantity (96 tests)
4 mL
100 µL
50 µL
8 mL
8 mL
50 µL
50 µL
50 µL
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Storage
Condition
(Before
Preparation)
4°C
-20°C
4°C
4°C
4°C
4°C
4°C
4°C
4°C
*Spin the solution down to the bottom prior to use.
These materials are not included in the kit, but will be required to successfully utilize this assay:
• Orbital shaker
• Pipettes and pipette tips
• Fluorescence microplate reader
• 1.5 mL microcentrifuge tubes
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GENERAL INFORMATION
 Assay kit intended for research use only. Not for use in diagnostic procedures
 Do not use kit or components if it has exceeded the expiration date on the kit labels
 Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted
 Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding
 Avoid foaming or bubbles when mixing or reconstituting components.
 Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.
 Ensure plates are properly sealed or covered during incubation steps.
 Complete removal of all solutions and buffers during wash steps.
 This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.
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ASSAY PREPARATION
Prepare fresh reagents immediately prior to use.
9.1
Completed HDM Assay Buffer
Prepare the Completed HDM Assay Buffer by adding HDM
Cofactor 1, 2, and 3, respectively into HDM Assay Buffer at a
1:100 ratio (e.g. add 1 μL of each Cofactor to 100 μL of HDM
Assay Buffer total volume 103 μL).
Prepare nuclear extracts by using you own successful method.
For your convenience and the best results, Abcam offers a Nuclear
Extraction Kit (ab113474) optimized for use with this kit.
Nuclear extracts can be used immediately or stored at –80°C for future use.
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ASSAY PROCEDURE
11.1 Determine the number of strip wells required. Leave these strips in the plate frame (remaining unused strips can be placed back in the bag. Seal the bag tightly and store at 4°C).
11.2
For the untreated control: Add 27 μL of Completed HDM Assay
Buffer, 2 μL of nuclear extract (5-10 μg) or purified enzyme, and 1 μL of HDM Substrate to the strip wells. Mix, cover the strip wells with
Parafilm M, and incubate at 37°C for 60 minutes.
11.3
For the blank: Add 2 μL of Completed HDM Assay Buffer instead of nuclear extract. For HDM inhibition, add 3 μL of the tested inhibitors at different amounts, and reduce Completed HDM Assay Buffer volume to 24 μL.
11.4
For generating the standard curve: dilute HDM Standard with
Completed HDM Assay Buffer at different concentrations (e.g. 12.5,
25, 50, 100, and 200 μM).
Add 26 μL of Completed HDM Assay
Buffer, 1 μL of HDM Substrate, followed by adding 3 μL of the diluted
HDM Standard into the wells.
Note: The inhibitor compound solution and nuclear extract should not have thiol-containing chemicals, such as DTT, GSH, and
2-mercaptoethanol, as the thiol-containing chemicals may interfere with the fluorometric determination .
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ASSAY PROCEDURE
Well Type Component
Amount/Well
(µL)
Control Wells
Inhibitor Wells
Standard Wells
Blank Wells
Completed Assay Buffer
Assay Buffer
Nuclear Extract
Completed Assay Buffer
Assay Buffer
Nuclear Extract
Inhibitor
Completed Assay Buffer
Assay Buffer
HDM Standard
Completed Assay Buffer
Assay Buffer
24
1
2
3
27
1
2
26
1
2
3
29
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11.5 Prepare the Fluoro-Development Solution by mixing Fluoro Developer and Fluoro Enhancer at a ratio 1:1 (e.g. 1 mL of Fluoro Developer +
1 mL of Fluoro Enhancer).
11.6 Add 140 μL of the Fluoro-Development Solution into the wells and incubate at room temperature for 15 - 20 minutes away from light.
Measure and read fluorescence on a fluorescence microplate reader at Ex/Em = 370/470 nm.
Note: If the strip well frame does not fit the fluorescence reader, transfer the solution to a standard 96-well microplate and read fluorescence at
Ex/Em = 370/470 nm.
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DATA ANALYSIS
Calculate HDM (H3K9) activity or inhibition. For simple calculation:
HDM activity (RFU/h/µg) = Treated (tested) sample RFU – blank RFU
Untreated (control) sample RFU – blank RFU
Inhibition % = Inhibitor Sample RFU – blank RFU
Untreated (control) sample RFU – blank RFU
) X 1000
For an accurate calculation, plot Delta RFU (sample RFU – blank RFU) value versus amount of HDM Standard and determine the slope as delta
RFU/ng. Calculate HDM (H3K9) activity using the following formula:
Activity (ng/h/µg) = Untreated Control RFU – blank RFU
Slope x reaction time (1h) x protein amount added (µg)
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RESOURCES
Problem
No Signal for the
Sample
Cause
The protein sample is not properly extracted.
The protein amount is added into well insufficiently.
The sample is not prepared from fresh cells or tissues.
Nuclear extracts are incorrectly stored.
Reagents are added incorrectly.
Incubation time and temperature is incorrect.
Absence of HDM
(H3K9) activity in the sample due to treatment.
Solution
Ensure the protein extraction protocol is suitable for nuclear protein extraction.
Ensure extract contains a sufficient amount of protein.
The nuclear extracts from frozen cells/tissue significantly lose enzyme activity.
Fresh sample should be used.
Ensure the nuclear extracts are stored at
–80°C.
Check if reagents are added in order and if any steps of the procedure have been omitted by mistake.
Ensure the incubation time and temperature described in the protocol is followed correctly.
N/A
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RESOURCES
Problem
High Background
Present for the
Blank
Cause
Contaminated by nuclear extracts or
HDM standard.
Overdevelopment
Solution
Ensure the well is not contaminated from adding the nuclear extract or HDM standard accidentally or from using HDM standard contaminated tips.
Decrease development time in step 11.6.
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RESOURCES
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RESOURCES
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