SimpleChIP® Stem Cell Master Regulator Assay Kit (#8980) Datasheet Without Images Cell Signaling Technology

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

Product Information

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.

Species Reactivity:

Human

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.

Product 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 M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

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.

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