EpiQuik™ HDAC10 Assay Kit

EPIGENTEK
Complete Solutions for Epigenetics
EpiQuik™ HDAC10 Assay Kit
(Colorimetric)
Base Catalog # P-4052
PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
The EpiQuik™ HDAC10 Assay Kit is diesnged for measuring HDAC10 protein amounts quantitatively
from fresh tissue and cultured cells of human and mouse.
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
© Epigentek Group Inc. All rights reserved. Products are for research use only.
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P-4052
EPIGENTEK
Complete Solutions for Epigenetics
KIT CONTENTS
Component
48 Assays
Cat. #P-4052-48
96 Assays
Cat. #P-4052-96
Storage
Upon Receipt
WB (10X Wash Buffer)
12 ml
25 ml
4°C
AB (Assay Buffer)
5 ml
10 ml
4°C
BB (Blocking Buffer)
10 ml
20 ml
4°C
CA (Capture Antibody, 200 µg/ml)*
13 µl
26 µl
4°C
DA (Detection Antibody, 200 µg/ml)*
6 µl
10 µl
–20°C
ES (Enhancer Solution)*
6 µl
10 µl
–20°C
DS (Developing Solution)
6 ml
12 ml
4°C
SS (Stop Solution)
6 ml
11 ml
RT
HDAC10 control (100 µg/ml)*
8 µl
16 µl
–20°C
8-Well Assay Strips (With Frame)
6
12
4°C
User Guide
1
1
RT
*For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.
SHIPPING & STORAGE
The kit is shipped in two parts: one part at ambient room temperature, and the second part on frozen
ice packs at 4°C.
Upon receipt: (1) Store DA, ES, and HDAC10 Control at –20°C away from light; (2) Store WB, AB,
BB, CA, DS, and the 8-Well Assay Strips at 4°C away from light; (3) Store all other components at
room temperature. The kit is stable for up to 6 months from the shipment date, when stored properly.
Note: (1) Check if wash buffer, WB, contains salt precipitates before using. If so, warm (at room
temperature or 37°C) and shake the buffer until the salts are re-dissolved; (2) check if a blue color is
present in DS (Developing Solution), which would indicate contamination of the solution and should not
be used. To avoid contamination, transfer the amount of DS required into a secondary container (tube
or vial) before adding DS into the assay wells.
MATERIALS REQUIRED BUT NOT SUPPLIED

Adjustable pipette or multiple-channel pipette

Multiple-channel pipette reservoirs

Aerosol resistant pipette tips

Microplate reader capable of reading absorbance at 450 nm

1.5 ml microcentrifuge tubes

Incubator for 37°C incubation
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
© Epigentek Group Inc. All rights reserved. Products are for research use only.
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EPIGENTEK
Complete Solutions for Epigenetics

Distilled water

Nuclear extracts or purified HDAC10 enzyme

Parafilm M or aluminum foil
GENERAL PRODUCT INFORMATION
Quality Control: Each lot of the EpiQuik™ HDAC10 Assay Kit is tested against predetermined
specifications to ensure consistent product quality. Epigentek guarantees the performance of all
products in the manner described in our product instructions.
Product Warranty: If this product does not meet your expectations, simply contact our technical
support unit or your regional distributor. We also encourage you to contact us if you have any
suggestions about product performance or new applications and techniques.
Safety: Suitable lab coat, disposable gloves, and proper eye protection are required when working
with this product.
Product Updates: Epigentek reserves the right to change or modify any product to enhance its
performance and design. The information in this User Guide is subject to change at any time without
notice. Thus, only use the User Guide that was supplied with the kit when using that kit.
Usage Limitation: The EpiQuik™ HDAC10 Assay Kit is for research use only and is not intended for
diagnostic or therapeutic applications.
Intellectual Property: The EpiQuik™ HDAC10 Assay Kit and methods of use contain proprietary
technologies by Epigentek.
A BRIEF OVERVIEW
Histone deacetylases (HDACs) play a critical role in transcriptional repression of gene expression in
eukaryotic cells by catalyzing the hydrolytic removal of acetyl groups from histone lysine residues.
HDACs are tightly involved in cell cycle regulation, cell proliferation and in the development of human
cancer. HDAC inhibition displays significant effects on apoptosis, cell cycle arrest and differentiation in
cancer cells. HDAC inhibitors are currently being developed as potential anti-cancer agents. Three
distinct families of HDACs have been described, comprising a group of at least 20 proteins in humans.
HDAC10 is a class II histone deacetylase containing 669 amino acid residues. HDAC10 contains a
unique, putative second catalytic domain and resides in both the nucleus and cytoplasm. Data
suggests that HDAC10 may play roles both in the nucleus, as a transcriptional modulator, and in the
cytoplasm in an unidentified role. HDAC10 possesses histone deacetylase activity and represses
transcription through interaction with many transcription factors. HDAC10 regulates many biological
processes including cell cycle progression.
Western blot is currently the most prominent assay technique for measuring the expression or amount
of HDAC10 protein. Yet this traditional method requires electrophoresis and transfer processes, which
make the assay inconvenient, time consuming, and low throughput. The EpiQuik™ HDAC10 Assay Kit
addresses these problems by using a unique procedure to measure the amount of HDAC10 proteins.
The kit has the following features:

Very fast procedure, which can be finished within 4 hours.
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
© Epigentek Group Inc. All rights reserved. Products are for research use only.
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P-4052
EPIGENTEK
Complete Solutions for Epigenetics

Innovative colorimetric assay to quantitatively measure HDAC10 protein amount without the need
for electrophoresis.

High sensitivity and specificity – HDAC10-specific detection with a detection limit as low as 0.5 ng
of HDAC10 protein.

Strip microplate format makes the assay flexible: manual or high throughput analysis.

HDAC10 control is included, which allows for the HDAC10 protein amount of the sample to be
properly quantified.

Simple, reliable, and consistent assay conditions.
PRINCIPLE & PROCEDURE
The EpiQuik™ HDAC10 Assay Kit (Colorimetric) is designed for measuring total HDAC10 protein
amount from tissues or cells. In an assay with this kit, the unique HDAC affinity substrate is stably
coated on the strip well. The sample is added into the well and HDAC10 proteins contained in the
sample bind to the substrate. The bound HDAC10 can be recognized with a HDAC10-specific antibody
and colorimetrically quantified through an ELISA-like reaction. The amount of HDAC10 is proportional
to the intensity of color development.
Schematic procedure of the EpiQuik™ HDAC10 Assay Kit (Colorimetric)
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
© Epigentek Group Inc. All rights reserved. Products are for research use only.
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P-4052
EPIGENTEK
Complete Solutions for Epigenetics
0.6
0.5
OD450 nm
0.4
0.3
0.2
0.1
0
0
10
20
30
40
HDAC10 Control (ng)
Illustrated standard curve generated with HDAC10 control
ASSAY PROTOCOL
For the best results, please read the protocol in its entirety prior to starting your experiment.
Starting Materials
Input Amount: The amount of nuclear extracts for each assay can be between 0.5 µg and 10 ug with
an optimal range of 2 to 4 µg.
Nuclear Extraction: You can use your method of choice for preparing nuclear extracts from the treated
and untreated samples. Epigentek also offers a nuclear extraction kit (Cat # OP-0002) optimized for
use with this kit (see “Ordering Information”).
Nuclear extracts should be stored at –80°C in aliquots until use.
1. Working Buffer and Solution Preparation
a. Prepare Diluted WB 1X Wash Buffer:
48-Assay Kit: Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7.2-7.5.
96-Assay Kit: Add 26 ml of WB 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7.2-7.5.
This Diluted WB 1X Wash Buffer can now be stored at 4°C for up to six months.
b. Prepare Diluted CA Capture Antibody Solution:
Dilute CA Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1:200 (i.e., add 1 µl of CA to
200 µl of Diluted WB). 50 µl of Diluted CA will be required for each assay well.
c.
Prepare Diluted DA Detection Antibody Solution:
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
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Complete Solutions for Epigenetics
Dilute DA Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1:2000 (i.e., add 1 µl of DA
to 2000 µl of Diluted WB). 50 µl of Diluted DA will be required for each assay well.
d. Prepare Diluted ES Enhancer Solution:
Dilute ES Enhancer Solution with Diluted WB at a ratio of 1:5000 (i.e., add 1 µl of ES to 5000 µl of
Diluted WB). About 50 µl of this Diluted ES will be required for each assay well.
e. Prepare Diluted HDAC10 Control Standard:
Suggested Standard Curve Preparation: First, dilute HDAC10 control with AB to a concentration of 20
ng/µl by adding 2 µl of HDAC10 control to 8 µl of AB Then, further prepare concentration points of 1,
2, 5, 10 and 20 ng/µl according to the following chart:
Tube
HDAC10 (20
ng/µl)
AB
Resulting
HDAC10
Concentration
1
0.5 µl
9.5 µl
1 ng/µl
2
0.5 µl
4.5 µl
2 ng/µl
3
1.0 µl
3.0 µl
5 ng/µl
4
2.0 µl
2.0 µl
10 ng/µl
5
4.0 µl
0.0 µl
20 ng/µl
Note: Keep each of the diluted solutions except WB1X Wash Buffer on ice until use. Any remaining diluted
solutions other than Diluted WB should be discarded if not used within the same day. The lower
concentration point (ex: 0.5 ng/µl) can be also added if needed.
2. HDAC10 Binding
a.
Predetermine the number of strip wells required for your experiment. It is advised to run replicate
samples (include blank and positive controls) to ensure that the signal generated is validated. Carefully
remove un-needed strip wells from the plate frame and place them back in the bag (seal the bag tightly
and store at 4°C).
b.
Blank Wells: Add 100 µl of AB to each blank well.
c.
Standard Wells: Add 99 µl of AB and 1 µl of Diluted HDAC10 control to each standard well with a
minimum of five wells, each at a different concentration between 1 and 20 ng/µl (based on the dilution
chart in Step 1e; see Table 2 in the “Suggested Strip Well Setup” section as an example).
d.
Sample Wells: Add 94 to 98 µl of AB and 2 to 6 µl of your nuclear extracts to each sample well. Total
volume should be 100 µl per well.
Note: (1) Follow the diagram in the “Suggested Strip Well Setup” section; (2) It is recommended to use 2
µg to 4 µg of nuclear extract per well.
e.
Cover strip-well microplate with Parafilm M or aluminum foil to avoid evaporation and incubate at 37°C
for 90 to 120 min.
f.
Remove the reaction solution from each well. Add 150 µl of BB Blocking Buffer to each well, then
cover with Parafilm M or aluminum foil and incubate at 37°C for 30 min.
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
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EPIGENTEK
Complete Solutions for Epigenetics
g.
Remove the reaction solution from each well. Wash each well three times with 150 µl of the Diluted
WB 1X Wash Buffer each time.
3. Antibody Binding & Signal Enhancing
a.
Add 50 µl of the Diluted CA to each well, then cover with Parafilm M or aluminum foil and incubate at
room temperature for 60 min.
b.
Remove the Diluted CA solution from each well.
c.
Wash each well three times with 150 µl of the Diluted WB each time.
d.
Add 50 µl of the Diluted DA to each well, then cover with Parafilm M or aluminum foil and incubate at
room temperature for 30 min.
e.
Remove the Diluted DA solution from each well.
f.
Wash each well four times with 150 µl of the Diluted WB each time.
g.
Add 50 µl of the Diluted ES to each well, then cover with Parafilm M or aluminum foil and incubate at
room temperature for 30 min.
h.
Remove the Diluted ES solution from each well.
i.
Wash each well five times with 150 µl of the Diluted WB each time.
Note: Ensure any residual wash buffer in the wells is removed as much as possible at each wash step.
4. Signal Detection
a.
Add 100 µl of DS to each well and incubate at room temperature for 1 to 10 min away from light. Begin
monitoring color change in the sample wells and control wells. The DS solution will turn blue in the
presence of sufficient HDAC10 protein.
b.
Add 100 µl of SS to each well to stop enzyme reaction when color in the positive control wells turns
medium blue. The color will change to yellow after adding SS and the absorbance should be read on a
microplate reader within 2 to 10 min at 450 nm with an optional reference wavelength of 655 nm.
Note: (1) Most microplate readers have the capability to carry out dual wavelength analysis and will
automatically subtract reference wavelength absorbance from the test wavelength absorbance. If your plate
reader does not have this capability, the plate can be read twice, once at 450 nm and once at 655 nm. Then,
manually subtract the 655 nm ODs from 450 nm ODs; (2) If the strip-well microplate frame does not fit in the
microplate reader, transfer the solution to a standard 96-well microplate.
5. HDAC10 Calculation
a.
Calculate the average duplicate readings for the sample wells and blank wells.
b.
Calculate % HDAC10 change using the following formula:
Treated (Tested) Sample OD – Blank OD
HDAC10 change % =
x 100%
Untreated (Control) Sample OD – Blank OD
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
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EPIGENTEK
Complete Solutions for Epigenetics
Example calculation:
Average OD450 of treated sample is 0.5
Average OD450 of untreated control is 0.9
Average OD450 of blank is 0.1
HDAC10 change % =
(0.5 – 0.1)
x 100% = 50%
0.9- 0.1
For Detailed Quantification :
1.
Generate a standard curve and plot OD value versus amount of HDAC10 control standard at
each concentration point.
2.
Determine the slope as OD/ng (you can use Microsoft Excel statistical functions for slope
calculation), then calculate the amount of HDAC10 using the following formulas:
(Sample OD – Blank OD)
HDAC10 (ng/mg protein) =
x 1000
Slope x Protein Amount (ug*)
* Nuclear extract added into sample wells at Step 2d.
SUGGESTED BUFFER AND SOLUTION SETUP
Table 1. Approximate amount of required buffers and solutions for defined assay wells based on the protocol.
Reagents
1 well
1 strip
(8 wells)
2 strips
(16 wells)
6 strips
(48 wells)
12 strips
(96 wells)
Diluted WB
2.5 ml
20 ml
40 ml
120 ml
240 ml
AB
100 µl
800 µl
1600 µl
4900 µl
9600 µl
BB
0.15 ml
1.2 ml
2.5 ml
7.5 ml
14.5 ml
HDAC10 control
N/A
N/A
2 µL (optional)
4 µl
4 µl
Diluted CA
50 µl
400 µl
800 µl
2400 µl
4800 µl
Diluted DA
50 µl
400 µl
800 µl
2400 µl
4800 µl
Diluted ES
50 µl
400 µl
800 µl
2400 µl
4800 µl
DS
0.1 ml
0.8 ml
1.6 ml
4.8 ml
9.6 ml
SS
0.1 ml
0.8 ml
1.6 ml
4.8 ml
9.6 ml
SUGGESTED STRIP WELL SETUP
Table 2. The suggested strip-well plate setup for HDAC10 quantification in a 48-assay format (in a 96-assay format, Strips 7 to 12 can be
configured as Sample). The controls and samples can be measured in duplicate.
Well #
A
B
C
Strip 1
Blank
HDAC10 1 ng
HDAC10 2 ng
Strip 2
Blank
HDAC10 1 ng
HDAC10 2 ng
Strip 3
Sample
Sample
Sample
Strip 4
Sample
Sample
Sample
Strip 5
Sample
Sample
Sample
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Strip 6
Sample
Sample
Sample
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EPIGENTEK
Complete Solutions for Epigenetics
D
E
F
G
H
HDAC10 5 ng
HDAC10 10 ng
HDAC10 20 ng
Sample
Sample
HDAC10 5 ng
HDAC10 10 ng
HDAC10 20 ng
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
TROUBLESHOOTING
Problem
Possible Cause
Suggestion
No signal or weak signal
in both the positive
control and sample wells
Reagents are added incorrectly.
Check if reagents are added in the proper
order with the right amount, and if any
steps in the protocol may have been
omitted by mistake.
Incubation time and temperature
are incorrect.
Ensure the incubation time and
temperature described in the protocol are
followed correctly.
Incorrect absorbance reading.
Check if appropriate absorbance
wavelength (450 nm) is used.
Kit was not stored or handled
properly.
Ensure all components of the kit were
stored at the appropriate temperature and
the cap is tightly capped after each
opening or use.
The standard amount is
insufficiently added to the well in
Step 2c.
Ensure a sufficient amount of standard is
added.
The standard is degraded due to
improper storage conditions.
Follow the Shipping & Storage guidance
in this User Guide for storage of HDAC10
control.
Insufficient washing of wells.
Check if washing recommendations at
each step is performed according to the
protocol.
Contaminated by sample or
standard.
Ensure the well is not contaminated from
adding sample or standard accidentally or
from using contaminated tips.
Incubation time with Diluted DA is
too long.
The incubation time at Step 3d should not
exceed 90 min.
Over-development of color.
Decrease the development time in Step
4a before adding SS Stop Solution in
Step 4b.
Protein sample is not properly
extracted or purified.
Ensure your protocol is suitable for
histone protein extraction. For the best
results, it is advised to use Epigentek’s
Nuclear extraction Kit (Cat. No. OP-0002).
No signal or weak signal
in only the standard
curve wells
High background present
in the blank wells
No signal or weak signal
only in sample wells
110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
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EPIGENTEK
Complete Solutions for Epigenetics
Uneven color
development
Sample amount added into the
wells is insufficient.
Ensure a sufficient amount of nuclear
extracts is used as indicated in Step 2.
The sample can be titrated to determine
the optimal amount to use in the assay.
Sample was not stored properly or
has been stored for too long.
Ensure sample is stored in aliquots at –
80°C, with no more than 6 months nuclear
extracts.
Little or no HDAC10 in the sample.
This problem may be a result of many
factors. If the affecting factors cannot be
determined, use new or re-prepared
nuclear extracts.
Insufficient washing of the wells.
Ensure the wells are washed according to
the guidance of washing and residue
washing buffer is removed as much as
possible.
Delayed color development or
delayed stopping of color
development in the wells.
Ensure color development solution or stop
solution is added sequentially and is
consistent with the order you added the
other reagents (e.g., from well A to well G
or from well 1 to well 12).
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110 Bi County Blvd. Ste. 122, Farmingdale, NY 11735
Tel: 1-877-374-4368 ■ Fax: 1-718-484-3956 ■ E-mail: info@epigentek.com ■ Web: www.epigentek.com
© Epigentek Group Inc. All rights reserved. Products are for research use only.
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