EPIGENTEK

Complete Solutions for Epigenetics

QuantiSir™ Specific Gene Knockdown
Quantification Kit For CellDeath/Apoptosis
Base Catalog # P-5004

PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
The QuantiSir™ Specific Gene Knockdown Quantification Kit For Cell Death/Apoptosis is suitablefor quantifying gene knockdown caused by siRNA or antisense oligonucleotides using mammaliantissue and cell extracts.
The QuantiSir™ Specific Gene Knockdown Quantification Kit For Cell Death/Apoptosis series
offers a flexible choice of different kits used for measuring knockdown of 65 common genes
related to cell death/apoptosis.
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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KIT CONTENTS
Components

96 a ssays
P-5004-96

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

12 ml
28 ml
1 ml
20 ml
12 ml
10 ml
6 ml
5 µl
10 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 Detection Antibody at –20°C. Store Q2, Q4, Q6, GAPDH ControlAntibody, Capture Antibody, and 8-Well Assay Strips at 4°C away from light; (3) Store a ll 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 roomtemperature 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.

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P-5004

EPIGENTEK

Complete Solutions for Epigenetics

GENERAL PRODUCT INFORMATION
Qua lity Control: Epigentek guarantees the performance of all products in the manner described in
our 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 Cell
Death/Apoptosis is for research use only and is not intended for diagnostic or therapeuticapplication.
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 valuable technology for studying gene function. Gene knockdown leads to reduction inmRNA and subsequently protein expression. It can often be verified at mRNA level by northern blotor quantitative RT-PCR. However, decrease in the amount of a specific mRNA does not typicallycorrelate well with protein levels present in the cell. Gene knockdown can also be measured at theprotein level with western blot. Western blot analysis is the most comprehensive way of showingthat expression of the target gene has been downregulated. However this method, while sensitive,
often lacks the ability to discriminate between samples in which the differences in protein levels areminimal. 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 geneknockdown induced by siRNA or antisense oligonucleotide at the protein level in cultured cells ortissues. The assay system includes a general gene knockdown assay kit and the specific geneknockdown assay kits, and allows directly measuring a specific protein level in cell lysates. The kit
has the following features:
Quick and efficient procedure. Completion of 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 Cell Death/Apoptosis 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.

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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.

Schematic Procedure for Using theQuantiSir™ Specific Gene Knockdown
Quantification Kit For Cell
Death/Apoptosis

Quantification of HDAC1 knockdown.
MCF-7 cells were treated or untreatedwith HDAC1 siRNA. Protein extracts
were prepared and used for detectionof HDAC1 protein level.

PROTOCOL
Protein Ex tra ction
For Adherent Cells:
1. Grow cells (treated or untreated) to 70-80% confluency in 12 well or 6 well plate, trypsinize, andcollect cells into 15 ml tube.
2. Centrifuge the cells at 1000 rpm for 5 min and discard the supernatant. Add 1 ml of PBS into thecell pellet, suspend, and transfer cells into a 1.5 ml vial. Pellet cells again by centrifuging at 1000rpm for 5 min.
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, vortex, and incubate on ice for 10 min.
4. Pellet cell debris by centrifuging at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to anew vial. At this stage the supernatant can be used immediately or stored at –80°C.
Note: For 96 well plate cultures, Q1 can directly be added into the wells in 5 µl/well and incubate
at room temperature for 5 min to lyse cells. The lysed cell solution is transferred to a 0.5 ml vial
and centrifuge at 12,000 rpm for 10 min. Supernatant is transferred to a new 0.5 ml vial forstorage 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.

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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 min and discard the supernatant. Add 1 ml of PBS into thecell pellet, suspend and transfer cells into a 1.5 ml vial. Pellet cells again by centrifuging at 1000rpm for 5 min.
3. Remove supernatant as much as possible and add Q1 (50 µl/ 1 x 106 cells) to re-suspend cellpellet, vortex, and incubate on ice for 10 min.
4. Pellet cell debris by centrifuging at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to anew vial. At this stage the supernatant can be used immediately or stored at –80°C.
Ta rg et 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.
2. Dilute the protein extract with Q3 at a 1:1 ratio (ex: add 5 µl of Q3 to 5 µl of protein extracts).
Add 10 µl of the diluted protein extract into central area of each strip well. Spread out the solution
over the strip well surface by pipetting the solution up and down several times. Incubate the strip
wells at 37°C (with no humidity) for 90 min to evaporate the solution and dry the wells). For 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 min to dry the well.
3. Add 150 µl of Q4 to the wells and incubate at 37°C for 30-45 min.
4. Aspirate and wash the wells with 150 µl of d iluted Q2 three times.
5. Dilute GAPDH control antibody (at a 1:1000 ratio) to 1 µg/ml with Q5. Also dilute the capture
antibody (at a 1:500 ratio) to 1 µg/ml with Q5. Add 50 µl of the diluted GAPDH control antibody
and capture antibody to the wells and incubate at room temperature for 60 min on an orbital
shaker (50-100 rpm).
6. Aspirate and wash the wells with 150 µl of d iluted 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 min.
8. Aspirate and wash the wells with 150 µl of the d iluted Q2 five times.
9. Add 100 µl of Q6 to the wells and incubate at room temperature for 2-10 min away from light.
Monitor color development in the sample and control wells (blue).
10. Add 50 µl of Q7 to the wells and read absorbance on microplate reader at 450 nm.
11. Calculate % target protein level:
Protein % =

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

x 100%ODT (untreated control – blank)/ODC (treated sample – blank)
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.

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EPIGENTEK

Complete Solutions for Epigenetics

TROUBLESHOOTINGNo Signal for the Sample
The protein sample is not properly
extracted.

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

The protein amount is added into wellinsufficiently.

Ensure extract contains enough amount ofproteins.

Reagents are added incorrectly.

Check if reagents are added in order and if
any steps of the procedure may have been
omitted by mistake.

The well is not completely dried.

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

The well is incorrectly washed before
protein spotting.

Ensure the well is not washed before adding
protein extracts.

Incubation time and temperature
are incorrect.

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

Protein extracts are incorrectly stored.

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

Hig h Background Present for the Blank
The well is not washed enough.

Check if wash at each step is performed
according to the protocol.

Insufficient antibody dilution.

Increase antibody dilution.

Overdevelopment.

Decrease development time in step 9 of “targetprotein level detection.”

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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

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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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P-5004