Total CREB cellular kit
Simple and robust detection kit for Phospho CREB & Total CREB
Phospho-CREB (Ser133) cellular kit HTRF®
The phospho-CREB (S133) kit enables the cell-based quantitative detection of CREB when phosphorylated at Serine 133 as a readout of ERK pathway activation.
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Highly specific
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Ease-of-use
Overview
The phospho-CREB (S133) kit is optimal for the quantitative detection of the cyclic AMP-responsive element-binding protein when phosphorylated at S133, thus enabling the phosphorylation status of CREB to be checked with respect to its steady-state level in cells. CREB functions as a transcription factor, signaling downstream from MAPK/ERK and activated by several pathways, such as MAPK/ERK, PI3K/AKT and stress signaling. CREB plays a critical role in promoting neuronal plasticity, cognitive abilities, cell proliferation, survival, and glucose homeostasis.
Benefits
- COMPATIBLE WITH MANY CELL TYPES
- SPECIFICITY
Phospho-CREB (S133) assay principle
The Phospho-CREB (S133) assay measures CREB when phosphorylated at S133. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-CREB (S133) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.
Phospho-CREB (S133) 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-CREB (S133) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-CREB (S133) 1-plate assay protocol
Detection of Phosphorylated CREB (S133) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Glucose dose response: total and phospho-CREB assays on Min6 cells
Mouse pancreatic ß-cells Min6 are seeded in culture-treated 96-well plates at 70Kc/well and cultured for 6 days in complete culture medium at 37°C, 5% CO2. After a cell washing step with KRB (Krebs Ringer Buffer) and a quiescence step for 2h in KRB, cells were stimulated with 50 µL KRB and increasing concentrations of Glucose for 5 min at 37°C. After a 30 minutes lysis incubation time, total and phospho-CREB were measured using the two-plate assay protocol.
Forskolin Dose-Response on NIH 3T3 cells
Murine NIH 3T3 cells (50,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of forskolin. After a 30 minutes lysis incubation time, phosphorylated CREB was measured using the two-plate assay protocol.
Erlotinib Inhibition on A431 cells measured phospho-CREB kit
A431 cells (100,000 cells/well) were incubated for 30 min at 37°C with various concentrations of antagonist. Agonist (EGF) was then added and incubated for 10 minutes. After 30 minutes lysis incubation time, phosphorylated CREB was measured using the two-plate assay protocol.
HTRF versus WB phospho-CREB assays
8 million Hek293 cells were cultured and stimulated with 50 µM Forskolin. Following cell lysis, soluble fractions were then collected and centrifuged. Serial dilutions of cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were dispensed and analyzed side-by-side with Western Blot and HTRF. The HTRF phospho-CREB (Ser133) assay shows the same level of sensitivity as Western Blot.
CREB Simplified Pathway
CREB is a bZIP cellular transcription factor that activates transcription through cAMP response elements. CREB is able to mediate signals from numerous physiological stimuli, resulting in the regulation of a broad array of cellular responses. CREB plays a key role in promoting neuronal activity. Additionally, CREB signaling is involved in learning and memory, cell proliferation and survival, glucose homeostasis, spermatogenesis, and the circadian rhythm . CREB is activated by phosphorylation at Ser133 by various signaling pathways including Erk, Ca2+, and stress signaling. Some of the kinases involved in phosphorylating CREB at Ser133 are p90RSK, MSK, CaMKIV, PKA and MAPKAPK-2.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Changes in CREB and ERK phosphorylation levels after beta-amyloid treatment - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Cisbio lysis buffer compatibility
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
HTRF products and supporting scientific content to investifgate GPCRs - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
Multidimentional GPCR investigations - Posters
TCR signaling investigation with HTRF phospho assays
Study a pathway of interest in PBMC and T cells - Application Notes
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
HTRF and Tag-lite technologies in multidimensional cell-based assays for GPCR investigations
16 GPCR challenges, 1 solution - Posters
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Understanding GPCRs is the key to improved DD
In collaboration with GEN - Guides
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Product Insert CREB P-S133 Kit / 64CREPET
64CREPET - Product Insert
Product Insert CREB P-S133 Kit / 64CREPEG-64CREPEH
64CREPEG-64CREPEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
An innate and adaptive immunity recap
Insight into the diversity of cells & signaling pathways - Guides
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Innovative solutions in Virology research
Kits and reagents for virology research - Flyers
Safety Data Sheet (DEU) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (ELL) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (ITA) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (SPA) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) CREB P-S133 Kit / 64CREPEG
64CREPEG - Safety Data Sheet
Safety Data Sheet (DEU) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (ELL) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (ITA) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (SPA) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) CREB P-S133 Kit / 64CREPEH
64CREPEH - Safety Data Sheet
Safety Data Sheet (ELL) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (FRA-FR) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (ITA) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (SPA) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (ENG-GB) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (ENG-US) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (DEU) CREB P-S133 Kit / 64CREPET
64CREPET - Safety Data Sheet
Safety Data Sheet (DEU) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (ELL) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (ITA) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (SPA) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Safety Data Sheet (ENG-US) CREB P-S133 Kit / 64CREPEY
64CREPEY - Safety Data Sheet
Plate Reader Requirement
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