ab113458 – Histone H3 (K9) Activity Quantification Kit

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ab113458 –
Histone H3 (K9) Activity
Quantification Kit
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
Table of Contents
INTRODUCTION
1.
2.
BACKGROUND
ASSAY SUMMARY
1
2
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
3
3
4
4
6
6
ASSAY PREPARATION
9.
10.
REAGENT PREPARATION
SAMPLE PREPARATION
7
7
ASSAY PROCEDURE
11.
ASSAY PROCEDURE
8
DATA ANALYSIS
12.
ANALYSIS
10
RESOURCES
13.
14.
TROUBLESHOOTING
NOTES
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11
13
1
INTRODUCTION
1. BACKGROUND
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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2
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.
2. ASSAY SUMMARY
Nuclear Extract
Incubate with substrate and assay buffer
Add detection reagent
Fluorescence measurement
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3
GENERAL INFORMATION
3. PRECAUTIONS
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.
4. STORAGE AND STABILITY
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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4
GENERAL INFORMATION
5. MATERIALS SUPPLIED
Quantity (48
tests)
Quantity (96
tests)
HDM Assay Buffer
2 mL
4 mL
Storage
Condition
(Before
Preparation)
4°C
HDM Substrate
50 µL
100 µL
-20°C
HDM Standard, 10 mM*
25 µL
50 µL
4°C
Fluoro Developer*
4 mL
8 mL
4°C
Item
Fluoro Enhancer*
4 mL
8 mL
4°C
HDM Cofactor 1*
25 µL
50 µL
4°C
HDM Cofactor 2*
25 µL
50 µL
4°C
HDM Cofactor 3*
25 µL
50 µL
4°C
6
12
4°C
8-Well Assay Strip (with Frame)
*Spin the solution down to the bottom prior to use.
6. MATERIALS REQUIRED, NOT SUPPLIED
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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5
GENERAL INFORMATION
7. LIMITATIONS

Assay kit intended for research use only.
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
Not for use in diagnostic
8. TECHNICAL HINTS

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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6
ASSAY PREPARATION
9. REAGENT 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).
10. SAMPLE PREPARATION
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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7
ASSAY PROCEDURE
11. 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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8
ASSAY PROCEDURE
Well Type
Control Wells
Inhibitor Wells
Standard Wells
Component
(µL)
Completed Assay Buffer
27
Assay Buffer
1
Nuclear Extract
2
Completed Assay Buffer
24
Assay Buffer
1
Nuclear Extract
2
Inhibitor
3
Completed Assay Buffer
26
1
2
3
Assay Buffer
HDM Standard
Blank Wells
Amount/Well
Completed Assay Buffer
Assay Buffer
29
1
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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9
DATA ANALYSIS
12. 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 % =
1–(
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
13. TROUBLESHOOTING
Problem
Cause
Solution
No Signal for the
Sample
The protein sample is
not properly extracted.
Ensure the protein
extraction protocol is
suitable for nuclear
protein extraction.
The protein amount is
added into well
insufficiently.
Ensure extract
contains a sufficient
amount of protein.
The sample is not
prepared from fresh
cells or tissues.
The nuclear extracts
from frozen
cells/tissue
significantly lose
enzyme activity.
Fresh sample should
be used.
Nuclear extracts are
incorrectly stored.
Ensure the nuclear
extracts are stored at
–80°C.
Reagents are added
incorrectly.
Check if reagents are
added in order and if
any steps of the
procedure have been
omitted by mistake.
Incubation time and
temperature is
incorrect.
Ensure the
incubation time and
temperature
described in the
protocol is followed
correctly.
Absence of HDM
(H3K9) activity in the
sample due to
treatment.
N/A
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11
RESOURCES
Problem
Cause
Solution
High Background
Present for the
Blank
Contaminated by
nuclear extracts or
HDM standard.
Ensure the well is not
contaminated from
adding the nuclear
extract or HDM
standard accidentally
or from using HDM
standard
contaminated tips.
Overdevelopment
Decrease
development time in
step 11.6.
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RESOURCES
14. NOTES
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RESOURCES
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Japan
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All information / detail is correct at time of going to print.
RESOURCES
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