Revision 1

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Antibodies Included

Quantity

Application

Dilution

Oct-4A (C30A3C1) Rabbit mAb (ChIP Formulated) #5677

10 immunoprecipitations

ChIP

1:50

Sox2 (D6D9) XP® Rabbit mAb (ChIP Formulated) #5024

10 immunoprecipitations

ChIP

1:50

Nanog (D73G4) XP® Rabbit mAb (ChIP Formulated) #5232

10 immunoprecipitations

ChIP

1:50

Primers Included

Quantity

Application

Dilution

SimpleChIP® Human Oct-4 Promoter Primers #4641

250 PCR reactions

ChIP

1:10

SimpleChIP® Human α Satellite Repeat Primers #4486

250 PCR reactions

ChIP

1:10

Applications:
ChIP

Reactivity:
H

Sensitivity:
Endogenous

Product Usage Information

Directions for Use:A. Chromatin Immunoprecipitation:ChIP formulated antibodies have been tested and optimized using the SimpleChIP® Enzymatic Chromatin IP Kits (#9002 and #9003). Antibodies should be used at a dilution of 1:50 in a 500 μl ChIP reaction containing 10 to 15 μg of chromatin (4x106 cells). For the SimpleChIP® Enzymatic Chromatin IP protocol, please see the web page for this product at www.cellsignal.com.B. Quantification of DNA by qPCR:1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of real-time PCR machine to be used. PCR reactions should be performed in duplicate and should include a tube with no DNA to control for contamination, and a serial dilution of a 2% total input chromatin DNA (undiluted, 1:5, 1:25, 1:125), which is used to create a standard curve and determine amplification efficiency.2. Add 2 μl of the appropriate ChIP DNA sample to each tube or well of the PCR plate.3. Prepare a master PCR reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of the master PCR reaction mix to each PCR reaction tube or well of the PCR plate.Reagent Volume for 1 PCR Reaction (20 μl)

Nuclease-free H2O 6 μl

5 μM SimpleChIP® Primers 2 μl

2X SYBR-Green Reaction Mix 10 μl

4. Start the following PCR reaction program:

a. Initial Denaturation 95°C for 3 min

b. Denaturation 95°C for 15 sec

c. Anneal and Extension *Primer-specific temp. for 60 sec

d. Repeat steps b and c for a total of 40 cycles.*65 °C Anneal/Extension for SimpleChIP® Human Oct-4 Promoter Primers*60 °C Anneal/Extension for SimpleChIP® Human α Satellite Repeat Primers5. Analyze quantitative PCR results using software provided with the real-time PCR machine.

Storage

Antibodies are supplied in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide, and should be stored at -20°C. Do not aliquot the antibodies.
Primers are supplied in nuclease-free water at a concentration of 5 μM and should be stored at -20°C.

Specificity/Sensitivity

Each antibody in the SimpleChIP® Stem Cell Master Regulator Assay Kit detects endogenous levels of its respective human protein. SimpleChIP® Human Oct-4 Promoter Primers contain a mix of forward and reverse PCR primers that are specific for amplification of a 99 base pair region of the human Oct-4 promoter. SimpleChIP® Human α Satellite Repeat Primers contain a mix of forward and reverse PCR primers that are specific for the amplification of a 182 base pair region of the human α satellite repeat element.

Source / Purification

Oct-4 (C30A3C1) XP® Rabbit mAb (ChIP Formulated) is produced by immunizing animals with recombinant protein specific to the amino terminus of human Oct-4A protein. Sox2 (D6D9) XP® Rabbit mAb (ChIP Formulated) is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly179 of human Sox2 protein. Nanog (D73G4) XP® Rabbit mAb (ChIP Formulated) is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of human Nanog protein.

Description

The SimpleChIP® Stem Cell Master Regulator Assay Kit contains ChIP-formulated antibodies and SimpleChIP® primers for the analysis of Oct-4, Sox2 and Nanog binding to target genes in human cells by chromatin immunoprecipitation (ChIP). The positive control SimpleChIP® Human Oct-4 Promoter Primers are provided for detection and quantification of Oct-4 promoter enrichment, as Oct-4 is a known target gene of Oct-4, Sox2 and Nanog proteins. The negative control SimpleChIP® Human α Satellite Repeat Primers allow for determination of background levels of enrichment. Antibodies and primers are tested and optimized for parallel use with the SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and SYBR® Green quantitative real-time PCR. The kit provides enough reagents for 10 ChIP assays per antibody and 250 PCR reactions per primer set.

Background

Embryonic stem cells are pluripotent cells derived from the inner cell mass (ICM) of the mammalian blastocyst. Pluripotent cells are capable of indefinite self-renewing expansion in culture and can differentiate into cell types of all three germ layers: endoderm, ectoderm and mesoderm. This pluripotent state is a property shared by embryonic stem (ES) cells, embryonic carcinoma and induced pluripotent stem (iPS) cells. Oct-4, Sox2 and Nanog are key transcriptional regulators that are highly expressed in pluripotent cells (1). Together they form a master transcriptional regulatory network that maintains cells in a pluripotent state (2,3). Over-expression of Oct-4 and Sox2 along with Klf4 and c-Myc can induce pluripotency in both mouse and human somatic cells, highlighting their roles as key regulators of the transcriptional network necessary for self-renewal and pluripotency (4,5). It has also been demonstrated that overexpression of Oct-4, Sox2, Nanog and Lin28 can induce pluripotency in human somatic cells (6). Chromatin immunoprecipitation (ChIP) is a powerful technique that can be used to identify Oct-4, Sox2 and Nanog target genes in a given population of pluripotent cells (2,3,7-9). In addition, ChIP can be used to characterize changes in target gene occupancy that occur during induction of iPS cells from somatic cells, or differentiation of pluripotent cells into different cell lineages.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Applications Key

ChIP: Chromatin IP

Cross-Reactivity Key

H: Human

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 NCCIT cells and 10 μl of Nanog, Oct-4 and Sox2 antibodies or 2 μl of Normal Rabbit IgG, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human Nanog promoter primers, SimpleChIP® Human Oct-4 Promoter Primers #4641, SimpleChIP® Human Sox2 Promoter Primers #4649, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Chromatin Immunoprecipitation Image 1: SimpleChIP<sup>®</sup> Stem Cell Master Regulator Assay Kit
NTERA-2 cells were either untreated (left panel) or treated for 15 days with retinoic acid (RA) to induce differentiation along the neuronal lineage (right panel). Chromatin immunoprecipitations were then performed with cross-linked chromatin from 4 x 106 cells and 10 μl of Nanog, Oct-4, and Sox2 antibodies, or 2 μl of Normal Rabbit IgG, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human Nanog promoter primers, SimpleChIP® Human Oct-4 Promoter Primers #4641, SimpleChIP® Human Sox2 Promoter Primers #4649, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one. Note the loss of Nanog, Oct-4, and Sox2 binding to target genes as NTERA-2 cells are induced to differentiate.
Chromatin Immunoprecipitation Image 2: SimpleChIP<sup>®</sup> Stem Cell Master Regulator Assay Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.