EPIGENTEK
Complete Solutions for Epigenetics

QuantiSir™ Specific Gene Knockdown
Quantification Kit For EpigeneticRegulators
Base Catalog # P-5002

PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
The QuantiSir™ Specific Gene Knockdown Quantification Kit For Epigenetic Regulators is suitablefor quantifying gene knockdown caused by siRNA or antisense oligonucleotides using mammaliantissue and cell extracts.
The QuantiSir™ Specific Gene Knockdown Quantification Kit For Epigenetic Regulators series
offers a flexible choice of different kits used for measuring knockdown of 37 common genes
related to epigenetic regulation.
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.

Page 1Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

KIT CONTENTS
Components

48 assays
P-5002-48

96 assays
P-5002-96

Q1 (Extraction Buffer)
Q2 (10X Wash Buffer)
Q3 (Protein Capture Buffer)
Q4 (Blocking Buffer)
Q5 (Antibody Buffer)
Q6 (Developing Solution)
Q7 (Stop Solution)
GAPDH Control Antibody*Capture Antibody*
Detection Antibody*
8-Well Assay Strips (with Frame)
User Guide

6 ml
14 ml
0.5 ml
10 ml
6 ml
5 ml
3 ml
5 µl
30 µl
10 µl
61

12 ml
28 ml
1 ml
20 ml
12 ml
10 ml
6 ml
10 µl
60 µl
20 µl
121

* 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 onfrozen ice packs at 4°C.
Upon receipt: (1) Store Capture Antibody and Detection Antibody at –20°C; (2) Store Q2, Q4,Q6, GAPDH Control Antibody, and 8-Well Assay Strips at 4°C away from light; (3) Store all othercomponents at room temperature. The kit is stable for up to 6 months from the shipment date,when stored properly.
Note: Check if wash buffer, Q2, contains salt precipitates before using. If so, warm (at room
temperature or 37°C) and shake the buffer until the salts are re-dissolved.

MATERIALS REQUIRED BUT NOT SUPPLIED Centrifuge
 Orbital shaker
 Microplate reader Pipettes and pipette tips
 15 conical tubes
 1.5 ml microcentrifuge tubes
 PBS
 Distilled water
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.

Page 2Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

GENERAL PRODUCT INFORMATION
Quality Control: Epigentek guarantees the performance of all products in the manner described inour product instructions.
Product Updates: Epigentek reserves the right to change or modify any product to enhance its
performance and design.
Usage Limitation: The QuantiSir™ Specific Gene Knockdown Quantification Kit For EpigeneticRegulators is for research use only and is not intended for diagnostic or therapeutic application.
Intellectual Property: The QuantiSir™ kits and methods of use contain proprietary technologies byEpigentek. QuantiSir™ is a trademark of Epigentek, Inc.

A BRIEF OVERVIEW
Targeted gene knockdown using small interfering RNA (siRNA) or antisense oligonucleotide has
been important technology for studying gene function. Gene knockdown leads to reduction inmRNA and subsequently protein expression. It can be often verified at mRNA level by Northernblot or quantitative RT-PCR. However, a decrease in the amount of a specific mRNA does not
typically correlate well with protein levels present in the cell. Gene knockdown can also bemeasured at the protein level with Western blot. Western blot analysis is the most comprehensive
way of showing that expression of the target gene has been down-regulated. However, this method
while sensitive, often lacks the ability to discriminate between samples in which the differences inprotein levels are minimal. It is also limited in its application to high-throughput analysis. To
address these problems, Epigentek has developed the QuantiSir™ gene knockdown assay system
to quantify gene knockdown induced by siRNA or antisense oligonucleotide at the protein level incultured cells or tissues. The assay system includes a general gene knockdown assay kit and thespecific gene knockdown assay kits, and allows directly measuring a specific protein level in celllysates. The kit has the following features:Quick and efficient procedure – completion of the entire assay needs only 4 hours.

Innovative colorimetric assay without the need for radioactivity, electrophoresis, orchromatography.

The convenient internal control is included to correct for the variations for the cell number orprotein concentrations.

Strip microplate format makes the assay flexible: manual or high throughput.

Simple, reliable, and consistent assay conditions.

PRINCIPLE & PROCEDURE
The QuantiSir™ Specific Gene Knockdown Quantification Kit For Epigenetic Regulators isspecifically designed for quantifying gene knockdown induced by siRNA or antisense
oligonucleotide at the protein level in the cultured cells or tissues. In the assay, the cell lysatescontaining the targeted protein are stably spotted on the specifically treated microwells with unique

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.

Page 3
Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

protein capture buffer. The spotted protein can then be recognized with the target-specific antibody
and colorimetrically measured through detection antibody-chromogen reaction system.

0.4
controlknockdow n

Delta OD

0.3

0.2

0.1

01
GAPDH

Schematic Procedure for Using the QuantiSir™ Specific Gene
Knockdown Quantification Kit For Epigenetic Regulators

2HDAC1

Quantification of HDAC1 knockdown. MCF-7
cells were treated or untreated with HDAC1siRNA. Protein extracts were prepared andused for detection of HDAC1 protein level.

PROTOCOL
Protein Extraction
For Adherent Cells:
1. Grow cells (treated or untreated) to 70-80% confluency in 12-well or 6- well plate, trypsinize, andcollect cells into a 15 ml tube.
2. Centrifuge the cells at 1000 rpm for 5 minutes and discard the supernatant. Add 1 ml of PBS into
the cell pellet, suspend, and transfer cells into a 1.5 ml vial. Pellet cells again by centrifuging at
1000 rpm for 5 minutes.
3. Remove supernatant as much as possible and add Q1 (40 µl/well for 12- well plate and 100µl/well for 6-well plate) to re-suspend cell pellet, then vortex and incubate on ice for 10 minutes.
4. Pellet cell debris by centrifuging at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant toa new vial; at this stage the supernatant can be immediately used or store at –80°C.
Note: For 96-well plate cultures, Q1 can be directly added into the wells in 5 µl/well and incubatedat room temperature for 5 minutes to lyse cells. The lysed cell solution is transferred to a 0.5 ml vial
and centrifuged at 12,000 rpm for 10 minutes. Supernatant is then transferred to a new 0.5 ml vialfor storage or to the strip well for assay (see below).
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.

Page 4
Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

For Suspension Cells:
1. Collect cells (treated or untreated) into a 15 ml conical tube. Count cells in a hemacytometer.
2. Centrifuge the cells at 1000 rpm for 5 minutes and discard the supernatant. Add 1 ml of PBS into
the cell pellet, suspend and transfer cells into a 1.5 ml vial. Pellet cells again by centrifuging at
1000 rpm for 5 minutes.
3. Remove supernatant as much as possible and add Q1 (50 µl/1 x 106 cells) to re-suspend cellpellet, then vortex and incubate on ice for 10 minutes.
4. Pellet cell debris by centrifuging at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant toa new vial; at this stage the supernatant can be immediately used or store at –80°C.
Target Protein Level Detection
1. Determine the number of strip wells required. Leave these strips in the plate frame (remainingunused strips can be placed back in the bag. Seal the bag tightly and store at 4°C). Dilute Q2 with
distilled water (pH 7.2-7.5) at a 1:10 ratio (ex: 1 ml of Q2 + 9 ml of distilled water).
2. Dilute the protein extract with Q3 at a 1:1 ratio (ex: 5 µl of Q3 + 5 µl of protein extracts). Add 10µl of the diluted protein extract into the central area of each strip well. Spread out the solution overthe strip well surface by pipetting the solution up and down several times. Incubate the strip wells at37°C (with no humidity) for 90 minutes to evaporate the solution and dry the wells. For the blank,
add 10 µl of Q3 instead of protein extract.
Note: The non-evaporated solution may be gathered along the edges at the bottom of the well.
Make sure the well is completely dry by slightly tilting the well and aspirating against the edge witha P-10 or P-20 pipette. If there is still the residue solution, extend incubation time for an additional
15-30 minutes to dry the well.
3. Add 150 µl of Q4 to the wells and incubate at 37°C for 30-45 minutes.
4. Aspirate and wash the wells with 150 µl of diluted Q2 three times.
5. Dilute the GAPDH Control Antibody (at a 1:1000 ratio) to 1 µg/ml with Q5. Also dilute theCapture Antibody (at a 1:200 ratio) to 1 µg/ml with Q5. Add 50 µl of the diluted GAPDH ControlAntibody and diluted Capture Antibody, respectively to the wells and incubate at room temperaturefor 60 minutes on an orbital shaker (50-100 rpm).
6. Aspirate and wash the wells with 150 µl of diluted Q2 four times.
7. Dilute the Detection Antibody (at a 1:1000 ratio) with Q5. Add 50 µl of the diluted Detection
Antibody to each well. Incubate at room temperature for 30 minutes.
8. Aspirate and wash the wells with 150 µl of the diluted Q2 five times.
9. Add 100 µl of Q6 to the wells and incubate at room temperature for 2-10 minutes away fromlight. Monitor the color development in the sample and control wells (blue).
10. Add 50 µl of Q7 to the wells and read absorbance on a microplate reader at 450 nm.
11. Calculate % target protein level:
Protein % =

ODT (treated sample – blank)/ODC (untreated control – blank)
ODT (untreated control – blank)/ODC (treated sample – blank)

× 100%*Here ODT is OD value for the target protein. ODC is OD value for the GAPDH control.
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.

Page 5
Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

TROUBLESHOOTINGNo Signal for the Sample
The protein sample is notproperly extracted.

Ensure the protein extraction protocol issuitable for your protein sample preparation.

The protein amount is added
into well insufficiently.

Ensure extract contains a sufficient amount ofproteins.

Reagents are added incorrectly.

Check if reagents are added in the proper orderand if any steps of the procedure may havebeen omitted by mistake.

The well is not completely dried.

Ensure the well is incubated with no humidity anddry before adding the blocking buffer.

The well is incorrectly washedbefore protein spotting.

Ensure the well is not washed before
adding protein extracts.

Incubation time andtemperature are incorrect

Ensure the incubation time and temperaturedescribed in the protocol are correctly followed.

Protein extracts are incorrectlystored.

Ensure the protein extracts are stored at–80°C.

High Background Present for the Blank
The well is not washedsufficiently.

Check if wash at each step is performedaccording to the protocol.

Insufficient antibody dilution.

Increase antibody dilution.

Overdevelopment.

Decrease development time in step 9 of “Target
Protein Level Detection.”

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.

Page 6
Printed 2014-09-23
P-5002EPIGENTEK
Complete Solutions for Epigenetics

RELATED PRODUCTS
Target

DNMT1
Dnmt2
Dnmt3aDnmt3bMBD2MeCP2HDAC1HDAC2HDAC3HDAC4HDAC5HDAC6HDAC7HDAC8HDAC9HDAC10HDAC11
SIRT1LSD1Cat. No.

5002-DNMT15002-DNMT25002-DNMT3A5002-DNMT3B
5002-MBD25002-MECP25002-HDAC15002-HDAC25002-HDAC35002-HDAC45002-HDAC55002-HDAC65002-HDAC75002-HDAC85002-HDAC95002-HDAC105002-HDAC115002-SIRT15002-LSD1Target

SUMO1SUMO2NCOR2
HAT1P300

Cat. No.

5002-SUMO15002-SUMO25002-NCOR25002-HAT15002-P300
RNA Polymerase II 5002-RPII
SRC15002-SRC1SUV39H15002-SUV39H1TFIIB
5002-TFIIBP-130CAF15002-P130CAF1EED5002-EEDPARP
5002-PARP
G9a5002-G9A
SETDB15002-SETDB1
EZH25002-EZH2
SET95002-SET9
NSD15002-NSD1
TAFII2505002-TAFII250

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.

Page 7Printed 2014-09-23P-5002