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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  • Ease-of-use Ease-of-use
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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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 protein’s phosphorylation state under a no-wash assay format.
Phospho-CREB (S133) assay principle

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

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.
Phospho-CREB (S133) 2-plate assay protocol

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.

Glucose dose response: total and phospho-CREB assays on Min6 cells

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.

Forskolin Dose-Response on NIH 3T3 cells

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.

Erlotinib Inhibition on A431 cells measured phospho-CREB kit

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.
Western Blot versus HTRF phospho-CREB assays
Western Blot versus HTRF phospho- and total CREB assays

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.
CREB signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Analysis of the effect of aggregated beta-Amyloid on cellular signaling pathways critical for memory in Alzheimer's disease

Changes in CREB and ERK phosphorylation levels after beta-amyloid treatment - Application Notes

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

GPCR research from A to Z

HTRF products and supporting scientific content to investifgate GPCRs - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - 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

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

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 / 64CREPEG-64CREPEH

64CREPEG-64CREPEH - Product Insert

Cisbio Product Catalog 2019

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

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