Product Pathways - Chromatin Regulation / Epigenetics
SimpleChIP® Stem Cell Master Regulator Assay Kit #8980
|8980S||1 Kit (10 immunoprecipitations)||---||In Stock||---|
|8980||carrier free and custom formulation / quantity||email request|
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|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|
|SimpleChIP® Human Oct-4 Promoter Primers #4641||250 PCR reactions||ChIP|
|SimpleChIP® Human α Satellite Repeat Primers #4486||250 PCR reactions||ChIP|
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.
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.
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.
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.
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.
- Looijenga, L.H. et al. (2003) Cancer Res 63, 2244-50.
- Boyer, L.A. et al. (2005) Cell 122, 947-56.
- Loh, Y.H. et al. (2006) Nat Genet 38, 431-40.
- Takahashi, K. and Yamanaka, S. (2006) Cell 126, 663-76.
- Okita, K. et al. (2007) Nature 448, 313-7.
- Yu, J. et al. (2007) Science 318, 1917-20.
- Okumura-Nakanishi, S. et al. (2005) J Biol Chem 280, 5307-17.
- Catena, R. et al. (2004) J Biol Chem 279, 41846-57.
- Rodda, D.J. et al. (2005) J Biol Chem 280, 24731-7.
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For Research Use Only. Not For Use In Diagnostic Procedures.
SYBR® Green is a registered trademark of Molecular Probes, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, Inc.