Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure phosphorylated human, mouse, and rat CREB (S133) in cell lysates. An immobilized capture antibody specific for CREB binds both phosphorylated and unphosphorylated CREB. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15- (96-well) plate pack sizes
- Economical alternative to Western blot
Kit Content
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
- Streptavidin-HRP
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Scientific Data
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Specificity of Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA Shown by Peptide Competition. A lysate prepared from HeLa human cervical epithelial carcinoma cells treated with 200 nM PMA for 20 minutes was analyzed with this DuoSet IC ELISA. The Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC Detection Antibody was either untreated (no peptide) or pre-incubated with a phosphopeptide containing the CREB S133 phosphorylation site, a phosphopeptide containing the GSK-3 beta S9 phosphorylation site, a phosphopeptide containing the c-Jun S63 phosphorylation site, or a phosphopeptide containing the Raf1 S338 phosphorylation site. Peptides were used at 40 ng/mL. Only the phosphopeptide containing the CREB S133 phosphorylation site blocks the signal, indicating that the DuoSet IC ELISA is specific for CREB phosphorylation at S133.
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Quantification of Phosphorylated CREB in PMA-treated Human HeLa Cells. Lysates prepared from HeLa human cervical epithelial carcinoma cells either uninduced or induced with 200 nM PMA for the indicated times were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either Anti-Human Phospho-CREB (S133) (Catalog # AF2510) or Anti-Human CREB (Catalog # AF2989) Polyclonal Antibodies. The DuoSet IC ELISA results correlate well with the amounts of phosphorylated CREB detected by Western blot. The immunoblot with anti-CREB antibody indicates that total levels of human CREB remained constant during incubation with PMA.The quantification of phosphorylated CREB with this DuoSet IC ELISA was also determined using cells pretreated with two concentrations of the MEK inhibitor U0126, which indirectly inhibits phosphorylation of CREB at S133.
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Quantification of Phosphorylated CREB in U0126-treated HeLa Cells. HeLa human cervical epithelial carcinoma cells were untreated or treated with 200 nM PMA for 20 minutes, either with or without U0126 at the indicated concentrations. Cells were lysed and CREB phosphorylated at S133 was quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either anti-phospho-CREB (S133) or anti-CREB antibodies. The DuoSet IC ELISA results correlate well with the amounts of phosphorylated CREB detected by Western blot. The immunoblot with anti-CREB antibody indicates that total levels of human CREB remained constant during the various treatments.The Phospho-CREB (S133) DuoSet IC ELISA also quantifies phosphorylated CREB levels in mouse and rat cell lysates.
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Quantification of Phosphorylated CREB in Growth Factor-treated Mouse and Rat Cells. Lysates prepared from mouse NIH/3T3 cells either uninduced or induced with 10 ng/mL of human PDGF (Catalog # 120-HD) for 5 minutes (left panel), and rat PC12 cells either uninduced or induced with 100 ng/mL of recombinant rat beta-NGF (Catalog # 556-NG) for 10 minutes (right panel), were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with anti-phospho-CREB (S133) antibody. The DuoSet IC ELISA results correlate well with the amounts of phosphorylated CREB detected by Western blot.
Product Datasheets
Preparation and Storage
Background: CREB
The cAMP Response Element-Binding Protein (CREB) belongs to the bZIP superfamily of transcription factors, containing a basic domain that mediates DNA binding, and a leucine zipper domain that facilitates dimerization. Within the promoter of target genes, CREB dimers bind cAMP response elements, defined by the palindromic consensus sequence TGACGTCA. When phosphorylated at S133, CREB also binds the coactivator CREB binding protein (CBP), which enhances transcription by acetylating histones to facilitate chromatin unraveling.
Citations for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 9
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TSH stimulation of human thyroglobulin and thyroid peroxidase gene transcription is partially dependent on internalization
Authors: D Jang, E Eliseeva, J Klubo-Gwie, S Neumann, MC Gershengor
Cellular Signalling, 2021-12-09;90(0):110212.
Species: Human
Sample Types: Cell Lysates
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Adrenomedullin increases cAMP accumulation and BDNF expression in rat DRG and spinal motor neurons
Authors: M Sisakht, Z Khoshdel, A Mahmoodazd, SM Shafiee, MA Takhshid
Iranian journal of basic medical sciences, 2021-07-01;24(7):978-985.
Species: Rat
Sample Types: Cell Lysates
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FOSL2 promotes VEGF-independent angiogenesis by transcriptionnally activating Wnt5a in breast cancer-associated fibroblasts
Authors: X Wan, S Guan, Y Hou, Y Qin, H Zeng, L Yang, Y Qiao, S Liu, Q Li, T Jin, Y Qiu, M Liu
Theranostics, 2021-03-05;11(10):4975-4991.
Species: Human
Sample Types: Cell Culture Supernates
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E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms
Authors: MK Jolly, KE Ware, S Xu, S Gilja, S Shetler, Y Yang, X Wang, RG Austin, D Runyambo, AJ Hish, S Bartholf D, JT George, RT Kreulen, MK Boss, AL Lazarides, DL Kerr, DG Gerber, D Sivaraj, AJ Armstrong, MW Dewhirst, WC Eward, H Levine, JA Somarelli
Mol. Cancer Res., 2019-03-12;0(0):.
Species: Human
Sample Types: Cell Lysates
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4-Hydroxynonenal-induced GPR109A activation elicits bipolar responses, G?i-mediated anti-inflammatory effects, and G??-mediated cell death
Authors: J Gautam, S Banskota, S Shah, JG Jee, E Kwon, Y Wang, DY Kim, HW Chang, JA Kim
Br. J. Pharmacol., 2018-05-22;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Iso-?-acids, bitter components of beer, prevent obesity-induced cognitive decline
Authors: T Ayabe, R Ohya, K Kondo, Y Ano
Sci Rep, 2018-03-19;8(1):4760.
Species: Mouse
Sample Types: Tissue Homogenates
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[6]-Gingerol, from Zingiber officinale, potentiates GLP-1 mediated glucose-stimulated insulin secretion pathway in pancreatic ?-cells and increases RAB8/RAB10-regulated membrane presentation of GLUT4 transporters in skeletal muscle to improve hyperglycemia in Lepr(db/db) type 2 diabetic mice
Authors: MB Samad, MNAB Mohsin, BA Razu, MT Hossain, S Mahzabeen, N Unnoor, IA Muna, F Akhter, AU Kabir, JMA Hannan
BMC Complement Altern Med, 2017-08-09;17(1):395.
Species: Mouse
Sample Types: Tissue Homogenates
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Real-time monitoring of Cisplatin-induced cell death.
Authors: Alborzinia H, Can S, Holenya P, Scholl C, Lederer E, Kitanovic I, Wolfl S
PLoS ONE, 2011-05-16;6(5):e19714.
Species: Human
Sample Types: Cell Lysates
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Microarray-based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis.
Authors: Holenya P, Kitanovic I, Heigwer F, Wolfl S
Proteomics, 2011-04-18;11(10):2129-33.
Species: Human
Sample Types: Recombinant Protein
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