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.
Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
R&D Systems | Catalog # DYC2510-2
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Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Assay Range
31.2-2000 pg/mL
Sample Type
Cell Lysates
Reactivity
Human, Mouse, Rat
Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA 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
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Product Summary for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
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 Specifications
Assay Format
96-well strip plate (sold separately)
Sample Volume Required
Cell lysates (100 µL)
Detection Method
Colorimetric ELISA - 450nm (TMB)
Conjugate
Biotin
Specificity
Label
HRP
Scientific Data Images for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
Standard Curve
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.
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.
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.
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.Kit Contents for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
- 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
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: CREB
Long Name
cAMP Response Element-binding Protein
Alternate Names
CREB1
Gene Symbol
CREB1
Additional CREB Products
Product Documents for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
For research use only
Citations for Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA
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Protocols
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
- ELISA Sample Preparation & Collection Guide
- ELISA Troubleshooting Guide
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- Quantikine HS ELISA Kit Principle, Streptavidin-HRP Polymer
- Sandwich ELISA (Colorimetric) – Biotin/Streptavidin Detection Protocol
- Sandwich ELISA (Colorimetric) – Direct Detection Protocol
- Troubleshooting Guide: ELISA
- View all Protocols, Troubleshooting, Illustrated assays and Webinars
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Associated Pathways
TGF-beta Signaling Pathways
VEGF - VEGF R2 Signaling Pathways
