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Product listing: SimpleChIP® Human CDKN1A Promoter Primers, UniProt ID P38936 #6449 to SimpleChIP® Human HoxA2 Promoter Primers, UniProt ID O43364 #5517

$108
250 PCR reactions
500 µl
SimpleChIP® Human CDKN1A Promoter Primers contain a mix of forward and reverse PCR primers that are specific to the human CDKN1A promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The CDKN1A gene (also known as p21, CIP1 or WAF1) is an inhibitor of the cell cycle and is upregulated by p53 upon DNA damage.
REACTIVITY
Human

Background: The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

$122
20 µl
$293
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Western Blotting

Background: TNFRSF8/CD30 is a type-I transmembrane glycoprotein that is a member of the TNFR superfamily. CD30 is synthesized as a precursor protein that undergoes extensive posttranslational modification before becoming embedded in the plasma membrane as a 120-kDa transmembrane protein (1,2). The expression of CD30 is upregulated in activated T-cells and may trigger costimulatory signaling pathways upon its engagement (3,4). While its expression is normally restricted to subsets of activated T-cells and B-cells, CD30 expression is robustly upregulated in hematologic malignancies, such as Hodgkin’s lymphoma (HL), anaplastic large cell lymphoma (ALCL), and adult T-cell leukemia, thus making it an attractive target for therapeutic intervention (5,6). Research studies have suggested that in certain disease contexts, CD30 recruits TRAF2 and TRAF5 adaptor proteins to drive NF-kappa B activation, aberrant cell growth, and cytokine production (7-9). CD30 signaling is also regulated by TACE-dependent proteolytic cleavage of its ectodomain, which results in reduced CD30L-dependent activation of CD30+ cells (10, 11).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Western Blotting

Background: B-cell CLL/lymphoma 9-like protein (BCL9L, BL2, Bcl9-2, DLNB11) is a transcriptional activator that was originally identified in silico based on homology to BCL9 (1). BCL9L was subsequently found to play an important role in Wnt/β-catenin signaling by interacting with β-catenin and enhancing the transactivation potential of the β-catenin/TCF complex (2). Research studies show that BCL9L can increase the tumorigenic effect of aberrant Wnt signaling in some cases of colorectal cancer (2). Expression of BCL9L is correlated with tumor progression in colorectal (3) and breast cancer (4). Targeted deletion of BCL9 and BCL9L in the intestinal epithelium resulted in abrogation of Wnt target genes, including those controlling epithelial-mesenchymal transition and stem-cell like properties (5).

$262
100 transfections
300 µl
SignalSilence® Rheb siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Rheb expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: Ras Homolog Enriched in Brain (Rheb) is an evolutionarily conserved member of the Ras family of small GTP-binding proteins originally found to be rapidly induced by synaptic activity in the hippocampus following seizure (1). While it is expressed at relatively high levels in the brain, Rheb is widely expressed in other tissues and may be induced by growth factor stimulation. Like other Ras family members, Rheb triggers activation of the Raf-MEK-MAPK pathway (2). Biochemical and genetic studies demonstrate that Rheb has an important role in regulating the insulin/TOR signaling pathway (3-6). The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that acts as a sensor for ATP and amino acids, balancing the availability of nutrients with translation and cell growth. The tuberin/hamartin (TSC2/TSC1) complex inhibits mTOR activity indirectly by inhibiting Rheb through the tuberin GAP activity (7).

Each control slide contains formalin-fixed and paraffin-embedded SK-BR-3 cells, untreated and EGF-treated, that can serve as a control for immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify treatment efficacy.To be used with antibodies: 3777,4407, 4267, 2243, 4290, 2165, 2242, 4791.
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The 20S proteasome is the major proteolytic enzyme complex involved in intracellular protein degradation. PA700, PA28, and PA200 are three major protein complexes that function as activators of the 20S proteasome. There are three evolutionarily conserved subunits of PA28: PA28α (PSME1), PA28β (PSME2), and PA28γ (PSME3) (1,2). PA28α and PA28β form a heteroheptameric complex and function by binding to the 20S complex at its opening site(s). The PA28α/β complex is present throughout the cell and participates in MHC class I antigen presentation by promoting the generation of antigenic peptides from foreign proteins (2). PA28γ exists in the form of a homoheptamer and is mainly located in the nucleus. The PA28γ complex exerts its function by binding and guiding specific nuclear target proteins to the 20S proteasome for further degradation (3,4).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The eukaryotic mRNA 3' poly(A) tail interacts with the 5' cap structure to regulate translation of mRNA transcripts (1). The eIF4G translation initiation factor associates with poly(A) binding protein (PABP) to circularize mRNA (1,2). Paip1 is a PABP interacting protein that binds eIF4A to stimulate translation (3). A second PABP binding protein, Paip2, inhibits translation by interfering with PABP function. Specifically, Paip2 reduces the affinity of PABP for poly(A) RNA and competes with Paip1 for PABP binding (1,2).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: Twinfilin is an actin monomer-binding protein found in all eukaryotes (1). Mammals have three isoforms. Twinfilin-1 and twinfilin-2a are expressed in most non-muscle cell types, whereas twinfilin-2b is the main isoform in adult heart and skeletal muscle (2). Twinfilins are composed of two ADF-homology domains connected by a 30 kDa linker region. All twinfilins have been shown to form a 1:1 complex with G-actin, but not F-actin (reviewed in 3). Twinfilin-1 was originally known as A6 protein tyrosine kinase and thought to be part of a novel class of protein kinases. However, the protein was renamed after further studies showed no evidence of tyrosine kinase activity (4). Twinfilin-1 helps to prevent the actin filament assembly by forming a complex with actin monomers and, in mammals, has been shown to cap the filament barbed ends. It has been suggested that this regulates cell motility (5). Suppression of twinfilin-1 has also been shown to slow lymphoma cell migration to lymph nodes (6).

$263
100 tests
500 µl
This Cell Signaling Technology antibody is conjugated to violetFluor™ 450 and tested in-house for direct flow cytometry analysis in human cells.
APPLICATIONS
REACTIVITY
Human

Application Methods: Flow Cytometry

Background: Cluster of Differentiation 4 (CD4) is a glycoprotein composed of an amino-terminal extracellular domain (four domains: D1-D4 with Ig-like structures), a transmembrane part and a short cytoplasmic tail. CD4 is expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages and dendritic cells, and plays an important role in the development and activation of T cells. On T cells, CD4 is the co-receptor for the T cell receptor (TCR), and these two distinct structures recognize the Antigen–Major Histocompatibility Complex (MHC). Specifically, the D1 domain of CD4 interacts with the β2-domain of the MHC class II molecule. CD4 ensures specificity of the TCR–antigen interaction, prolongs the contact between the T cell and the antigen presenting cell and recruits the tyrosine kinase Lck, which is essential for T cell activation (1).

$263
100 tests
500 µl
This Cell Signaling Technology antibody is conjugated to PE and tested in-house for direct flow cytometry analysis in human cells.
APPLICATIONS
REACTIVITY
Human

Application Methods: Flow Cytometry

Background: CD14 is a leucine-rich repeat-containing pattern recognition receptor with expression largely restricted to the monocyte/macrophage cell lineage (1). Research studies have shown that CD14 is a bacterial lipopolysaccharide (LPS) binding glycoprotein, expressed as either a GPI-linked membrane protein or a soluble plasma protein (2). LPS induces an upregulation of GPI-linked CD14 expression, which facilitates TLR4 signaling and macrophage activation in response to bacterial infection (3-5).

$229
100 µg
This Cell Signaling Technology antibody is conjugated to APC and tested in-house for direct flow cytometric analysis in mouse cells.
APPLICATIONS
REACTIVITY
Mouse

Application Methods: Flow Cytometry

Background: F4/80 (EMR1) is a heavily glycosylated G-protein-coupled receptor and is a well-established marker for mouse macrophages (1-3). Expression of F4/80 has also been observed in microglia and subset populations of dendritic cells (4).

$262
3 nmol
300 µl
SignalSilence® Acetyl-CoA Carboxylase 1 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Acetyl-CoA Carboxylase 1 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce protein expression by western analysis.
REACTIVITY
Human

Background: Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA (1). It is the key enzyme in the biosynthesis and oxidation of fatty acids (1). In rodents, the 265 kDa ACC1 (ACCα) form is primarily expressed in lipogenic tissues, while 280 kDa ACC2 (ACCβ) is the main isoform in oxidative tissues (1,2). However, in humans, ACC2 is the predominant isoform in both lipogenic and oxidative tissues (1,2). Phosphorylation by AMPK at Ser79 or by PKA at Ser1200 inhibits the enzymatic activity of ACC (3). ACC is a potential target of anti-obesity drugs (4,5).

$262
50-100 transfections
300 µl
SignalSilence® MEK2 siRNA from Cell Signaling Technology allows the researcher to specifically inhibit MEK2 expression using RNA interference, a method in which gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products are rigorously tested in-house and have been shown to reduce protein expression in specified cell lines.
REACTIVITY
Human

Background: MEK1 and MEK2, also called MAPK or Erk kinases, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation (1-3). Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221, located in the activation loop of subdomain VIII, by Raf-like molecules. MEK1/2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx (1-4). Constitutively active forms of MEK1/2 are sufficient for the transformation of NIH/3T3 cells or the differentiation of PC-12 cells (4). MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII.

$262
3 nmol
300 µl
SignalSilence® Met siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Met expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor) is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl, and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. The addition of a phosphate at cytoplasmic Tyr1003 is essential for Met protein ubiquitination and degradation (4). Phosphorylation at Tyr1234/1235 in the Met kinase domain is critical for kinase activation. Phosphorylation at Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Research studies have shown that altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon, and breast. Thus, investigators have concluded that Met is an attractive potential cancer therapeutic and diagnostic target (6,7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The Mitotic Checkpoint Complex (MCC), which contains Bub1, Bub1b, Bub3, Mad2, and Cdc20, controls chromosome segregation and monitors kinetochore-microtubule interactions (1). During mitosis, the MCC complex inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C), thereby preventing cells with unaligned chromosomes from prematurely entering anaphase (2). Research studies have shown that Bub1b and Bub1 kinases are mutated in several types of human malignancies including hematopoietic, colorectal, lung, and breast cancers (3). Biallelic mutations in Bub1b have been found in mosaic variegated aneuploidy syndrome and premature chromatid separation syndrome (4). Bub1b mouse germline knockouts are embryonic lethal with heterozygous animals displaying genetic instability, early aging phenotypes, and increased cancer susceptibility (5). Bub3 binds both Bub1 and Bub1b, facilitating their recruitment to kinetochores (6), and is required for functional microtubule-kinetochore interactions (7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: CD83 is a single-transmembrane protein with a calculated molecular weight (MW) of 23 kDa, but due to heavy and differential glycosylation, its apparent MW ranges from 23 to 70 kDa. CD83 is predominantly expressed on mature dendritic cells (DCs) and has been used as a DC activation/maturation marker as its increased expression is correlated with upregulation of HLA class II antigen expression on DCs (1-3). CD83 is also expressed at a low level on lymphocytes and is upregulated upon lymphocyte activation (4, 5). Thymic epithelial cells also express CD83, which is required for normal CD4+ T cell development (6, 7). CD83 is also expressed as a soluble form (sCD83) that can be found in serum of healthy adults (8). sCD83 has been shown to negatively regulate immune response by lymphocytes (9, 10).

$260
100 µl
APPLICATIONS
REACTIVITY
Bovine, Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: The NDK/NME/NM23 kinase family (encoded by the NME gene family) consists of at least eight distinct proteins that exhibit different cellular localization (1). Members of this group inhibit metastasis in a variety of tumor cell types (2). All NDK/NME/NM23 proteins possess nucleoside diphosphatase kinase (NDK) activity and catalyze the phosphorylation of nucleoside diphosphate to the corresponding nucleoside triphosphate to regulate a diverse array of cellular events (3). At least four classes of NDK biochemical activities have been described, including protein-protein interactions (4-6), regulation of GTP-binding protein function (7-9), DNA-associated activities (10,11), and histidine-dependent protein phosphotransferase activity (12). NDK/NME proteins participate in the regulation of a broad spectrum of cellular responses, including development, differentiation, proliferation, endocytosis, and apoptosis (13). Because of its role in metastasis suppression and oncogenesis, NDKA (NME1/NM23-H1) has been widely studied (14). NDKA (NM23-H1) and NDKB (NM23-H2) are encoded by adjacent NME1 and NME2 genes and share 90% sequence identity. Two serine residues (Ser122 and Ser144) on NDKA/NM23-H1 can be phosphorylated by AMPKα1, but only phosphorylation at Ser122 determines whether NDKA channels ATP to AMPKα1. This regulates AMPKα1 activity towards ACC1, an important regulator of fatty acid metabolism (15). Mutation of NDKB/NM23-H2 at Ser122 (S122P) in melanoma cells results in altered phosphoryl transfer activity (16).

$108
250 PCR reactions
500 µl
SimpleChIP® Human Sox2 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human Sox2 promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The Sox2 gene is highly active in stem cells and the promoter is bound by Oct-4, Sox2, and Nanog proteins.
REACTIVITY
Human

Background: The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

$260
100 µl
APPLICATIONS
REACTIVITY
Mouse, Rat

Application Methods: Western Blotting

Background: SynGAP is a synaptic GTPase-activating protein selectively expressed in the brain and found at higher concentrations specifically at excitatory synapses in the mammalian forebrain. SynGAP has a PH domain, a C2 domain, and a highly conserved RasGAP domain, which negatively regulates both Ras activity and its downstream signaling pathways. SynGAP interacts with the PDZ domains of SAP102, as well as PSD95, a postsynaptic scaffolding protein that couples SynGAP to NMDA receptors (1). SynGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Ser765 and Ser1123, among other sites (2,3). Phosphorylation of SynGAP results in stimulation of the GTPase activity of Ras, and PSD95 dependent CaMKII phosphorylation of SynGAP increases after transient brain ischemia (1,4). SynGAP is implicated in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking and plays an important role in synaptic plasticity (3,5). SynGAP is critical during neuronal development as mice lacking SynGAP protein die postnatally. Furthermore, SynGAP mutant mice have reduced long-term potentiation (LTP) and perform poorly in spatial memory tasks (6).

$320
100 µg
This peptide is used to block IGF-I Receptor β Antibody #3027 reactivity in dot blot protocols.
APPLICATIONS

Application Methods: Immunohistochemistry (Paraffin)

Background: Insulin-like growth factor 1 (IGF-1) is a small (7.65 kDa) growth factor that interacts with both the IGF-1 receptor and the insulin receptor to control cell growth and apoptosis. Release of this endocrine hormone from the liver is stimulated by growth hormone produced in the anterior pituitary (1). Circulating IGF-1 is typically bound to one of six known IGF binding proteins (IGF-BP) (2). At target cells, the IGF-1 ligand binds IGF receptors (or insulin receptors) leading to receptor autophosphorylation and activation (3). Activated receptors mediate downstream signaling pathways (including Akt and MAPK) that regulate cell proliferation, apoptosis, development and longevity. Altered expression or mutation of IGF-1 is associated with several human disorders, including type I diabetes and various forms of cancer (4). Recombinant human IGF-1 has been used in clinical trials as a potential therapeutic agent in the treatment of human diseases (5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Western Blotting

Background: Sigma non-opioid intracellular receptor 1 (SIGMAR1) is an endoplasmic reticulum (ER) membrane chaperone that forms raft-like microdomains on the ER, where it interacts with mitochondria at the mitochondria-associated ER membrane domain (MAM). At MAM, SIGMAR1 maintains proper ER-mitochondrion Ca2+ signaling, regulates mitochondria function, and enhances cellular survival upon ER stress (1-4). When activated, SIGMAR1 translocates to ER and plasma membrane, where it interacts with a plethora of membrane proteins, including ion channels, neurotransmitter receptors, and kinases. SIGMAR1 also modulates a variety of neuronal functions, such as neuronal excitability, neuroplasticity, neuroprotection, and neurorestoration (5-7). SIGMAR1 binds to many anti-psychotic drugs and it is implicated in addiction, pain, neurodegenerative diseases, and depression (8-11). Recently, mutations in the SIGMAR1 gene have been reported to be associated with amyotrophic lateral sclerosis (12,13). Besides its important roles in central nervous system and peripheral nervous system, SIGMAR1 also enhances cancer cell migration and invasion (14,15).

$108
250 PCR reactions
500 µl
SimpleChIP® Human HoxA1 Intron 1 Primers contain a mix of forward and reverse PCR primers that are specific to intron 1 of the human Homeobox A1 (HoxA1) gene. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The homeobox gene clusters are silenced by members of the polycomb family.
REACTIVITY
Human

Background: The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein (PEBP) family that associates with Raf-1 and the MEK and MAP kinases (1). RKIP has been shown to form a complex with Raf-1, MEK, and Erk (2). Although MEK and Erk can simultaneously bind RKIP, the association between Raf-1 and RKIP and that of RKIP and MEK are mutually exclusive. Thus, RKIP competitively disrupts the Raf-1-MEK complex and effectively terminates signal transmission from Raf-1 to MAP kinases (2). The inhibitory effect of RKIP on MAP kinase signaling is eliminated by PKC phosphorylation of RKIP at Ser153 (3). PKC phosphorylation on Ser153 also promotes the association of RKIP with GRK2, which prevents GRK2-dependent internalization of GPCR (4). RKIP also interacts with modules of the NF-κB pathway, including NF-κB-inducing kinase (NIK), TAK1, IKKα and IKKβ (5). These interactions antagonize cytokine-induced activation of the NF-κB pathway (5). Restoration of RKIP expression is associated with the inhibition of prostate cancer metastasis, implying that RKIP may be a potential clinical target as a suppressor of tumor metastasis through inhibition of vascular invasion (6).

$108
250 PCR reactions
500 µl
SimpleChIP® Human GATA-6 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human GATA-6 promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology. The GATA-6 gene promoter is bivalent in stem cells, containing both histone H3 tri-methyl Lys4 and tri-methyl Lys27 epigenetic marks. When the GATA-6 gene is activated during endoderm development, the promoter becomes monovalent, as the histone H3 tri-methyl Lys27 mark is removed and the tri-methyl Lys4 mark is retained.
REACTIVITY
Human

Background: The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Western Blotting

Background: WW-domain binding protein 2 (WBP2) is an adaptor protein first identified in a screen for proteins that interact with YAP1 (1). WBP2 was subsequently shown to bind other WW domain-containing proteins, including TAZ and NEDD4-like ubiquitin-protein ligases (2-5). There is strong evidence for a conserved functional role for WBP2 in the Hippo kinase tumor suppressor pathway. WBP2 interaction is required for the oncogenic properties of TAZ in breast cancer cells (4) and the growth-promoting properties of the YAP homolog Yorkie in Drosophila (6). In vitro studies have also suggested that WBP2 may act as a transcriptional co-activator of both estrogen and progesterone receptors (7); this interaction was shown to be dependent upon tyrosine phosphorylation of WBP2 by c-Src and c-Yes kinases (8).

$269
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: MGMT (O-6-methylguanine-DNA methyltransferase) is a DNA repair enzyme that participates in a suicide reaction that specifically removes methyl or alkyl groups from the O(6) position of guanine, restoring guanine to its normal form without causing DNA breaks (1). MGMT protects cells from alkylating toxins, and is an important factor in drug resistance to alkylating therapeutic agents (2,3). It is ubiquitously expressed in normal human tissues (4) and is overexpressed in many types of human tumors, but epigenetically silenced in other tumors. MGMT silencing is a marker associated with poor prognosis, but is a good predictive marker for response to alkylating agent chemotherapy (5).

$269
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunohistochemistry (Paraffin)

Background: Phosphoinositide-3,4,5-triphosphate (PtdIns(3,4,5)P3)-dependent Rac exchanger 1 (PREX1) is a Rac-specific GTP-exchange factor (GEF) regulated by heterotrimeric G-protein β/γ subunits and the lipid second messenger PtdIns(3,4,5)P3 (1-4). PREX1 contains two DEP (Dishevelled, Egl-10, and Pleckstrin homology) domains that coordinate heterotrimeric G-protein signaling. It also contains a Dbl-homology domain, which exhibits Rac-GEF activity, and PH and PDZ domains for interacting with upstream and downstream signaling components (1). Originally shown to modulate cellular migration of neutrophils by Rac2 activation (5-8), it is clear that PREX1 plays a broader role in modulating cell migration. PREX1 promotes metastasis of prostate cancer and melanoma cells, affects endothelial junction integrity, and is required for platelet generation and function (9-14). Research studies suggest that PREX1 plays an essential role in mediating ErbB-dependent signaling events in breast cancer by coordinating Rac activation in response to paracrine signals within the tumor microenvironment. Activation of PREX1 downstream of ErbB3 and EGFR chemokine receptors (CXCR4) promotes Rac activation, increased migration, proliferation, tumorigenesis, and metastasis in breast cancer cells (15,16). Consistent with this observation, deletion of PREX1 expression in mice results in resistance to melanoma metastasis (11). Expression of PREX1 in human tumors transplanted into mice inversely correlates with increased tumor progression and poor survival (15). Additional research studies suggest that PREX Rac-GEF activity is enhanced by phosphorylation in response to growth factors or hormones, and may require coincident dephosphorylation of two PH domain serine residues. The upstream kinases and precise regulatory mechanism remains elusive (15,17).

$114
10 western blots
150 µl
Nonphosphorylated Elk-1 Control Proteins: Bacterially expressed Elk1 fusion protein serves as a negative control. Supplied in SDS Sample Buffer.Phosphorylated Elk-1 Control Proteins: Bacterially expressed Elk1 fusion protein phosphorylated by the Erk2 enzyme serves as a positive control. Supplied in SDS Sample Buffer.
APPLICATIONS

Application Methods: Western Blotting

Background: The transcription factor Elk-1 is a component of the ternary complex that binds the serum response element (SRE) and mediates gene activity in response to serum and growth factors (1-3). Elk-1 is phosphorylated by MAP kinase pathways at a cluster of S/T motifs at its carboxy terminus; phosphorylation at these sites, particularly Ser383, is critical for transcriptional activation by Elk-1. Elk-1 appears to be a direct target of activated MAP kinase: (a) biochemical studies indicate that Elk-1 is a good substrate for MAP kinase; (b) the kinetics of Elk-1 phosphorylation and activation correlate with MAP kinase activity; (c) interfering mutants of MAP kinase block Elk-1 activation in vivo. Other studies have shown that Elk-1 (Ser383) is also a target of the stress-activated kinase SAPK/JNK (4,5).

$108
250 PCR reactions
500 µl
SimpleChIP® Human HoxA2 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human Homeobox A2 (HoxA2) promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The homeobox gene clusters are silenced by members of the polycomb family.
REACTIVITY
Human

Background: The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.