The Total CREB kit monitors cellular CREB expression level and can be used as a normalization assay for the phospho-CREB kits.
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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
The Total CREB kit monitors cellular CREB expression level and can be used as a normalization assay for the phospho-CREB kits.
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Overview

The Total CREB cellular assay monitors total CREB and is used as a normalization assay with our phospho-CREB kits. This kit is compatible with the buffers from the phospho-CREB kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations. 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
  • DATA NORMALIZATION

Total-CREB assay principle

The Total-CREB assay quantifies the expression level of CREB in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-CREB assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of CREB in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total-CREB assay principle

Total-CREB 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 Total-CREB HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-CREB 2-plate assay protocol

Total-CREB 1-plate assay protocol

Detection of total CREB 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.
Total-CREB 1-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

Validation of the total CREB assay on HEK293 cells

Human embryonic kidney cells HEK293 are seeded in culture-treated 96-well plates at different cell densities/well in complete culture medium and incubated for 24 hours at 37°C, 5% CO2. Cells were then lysed with 50 µL of supplemented Lysis Buffer #1 for 30 min at RT, after a 30 minutes lysis incubation time, total CREB was measured using the two-plate assay protocol. The assay presents a good sensitivity allowing the detection of total CREB on 25,000 cells (S/N = 5). The excellent linearity of the assay allows total CREB to be detect in cell densities varying from 25000 to 200,000 cells/well.
Validation of the total CREB assay on HEK293 cells

HTRF versus WB total 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 total-CREB assay is 8-fold more sensitive than the Western Blot and shows optimal correlation.
Western Blot versus HTRF 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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - 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

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

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 total Kit / 63ADK052PEG-63ADK052PEH

63ADK052PEG-63ADK052PEH - 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

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

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

Safety Data Sheet CREB total Kit / 63ADK052PEG

63ADK052PEG - Safety Data Sheet

Safety Data Sheet CREB total Kit / 63ADK052PEH

63ADK052PEH - Safety Data Sheet

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