Phospho-CREB (Ser133) & Total CREB Cellular Assay Kits

Phospho- and total CREB assays for monitoring CREB pathway

Cisbio's cell-based homogeneous HTRF® total CREB assay kit is designed for the quantitative detection of the cyclic AMP-responsive element-binding protein, enabling  the phosphorylation status of CREB to be checked with respect to its steady-state level in cells. Ideal for CREB normalization, the total CREB 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 activated by several pathways, such as MAPK/ERK, PI3K/AKT and stress signaling. This means the protein plays a key role in many biological processes, for example promoting neuronal plasticity, cognitive abilities, cell proliferation, survival and glucose homeostasis. The HTRF® total-CREB assay is an ideal tool for oncology, CNS, diabetes and inflammation research.

 

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

HTRF® - THE HOMOGENEOUS CELL-BASED SANDWICH IMMUNOASSAY

Cisbio Bioassay’s phospho- and total CREB assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-CREB antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with CREB, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-CREB can be quantitatively detected using the HTRF phospho-CREB (Ser133) and total CREB cellular assay kit reagents, and results obtained with most TR-FRET multimode plate readers.

A SIMPLER, MORE FLEXIBLE ASSAY PROTOCOL - ADAPTED TO YOUR APPLICATIONS

TWO-PLATE ASSAY PROTOCOL

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are first plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

ONE-PLATE ASSAY PROTOCOL

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, giving fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Guidelines and tips to get the best out of your assays

Guidelines for cell culture and Lysis to prior detection, Download tehcnical note

Download our cell-based phospho-protein data normalization application note

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.

Product Performance

1. Validation of total and phospho- CREB assays on Min6 cells: glucose dose-response

Mouse pancreatic β-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 minute lysis incubation time, total and phospho-CREB were measured using the two-plate assay protocol

2. FORSKOLIN DOSE-RESPONSE ON NIH 3T3 CELLS measured by the HTRF phospho-CREB cellular assay

Murine NIH 3T3 cells (50,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of forskolin. After a 30 minute lysis incubation time, phosphorylated CREB was measured using the two-plate assay protocol.

3. INHIBITION EFFECT OF ERLOTINIB on A431 cells measured by the HTRF phospho-CREB cellular assay

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 a 30 minute lysis incubation time, phosphorylated CREB was measured using the two-plate assay protocol.

4. 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 minute 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.

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

8 million Hek293 cells were grown in a T175 flask at 37°C, 5% CO2 for 48h in complete culture medium.

After washing with 10 mL PBS, samples were stimulated with 50 µM Forskolin for 30 min. After medium removal, and washing with 10 mL PBS, the cells were lysed with 3ml of supplemented lysis buffer #1 for 30 min at room temperature.

Soluble fractions were then collected after a 10 min centrifugation. 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 while

·The HTRF total- CREB is 8-fold more sensitive than the Western Blot and shows optimal correlation

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
CREB total kit - 500 tests63ADK052PEG
CREB total kit - 10,000 tests63ADK052PEH
CREB total kit - 50,000 tests63ADK052PEY
-
CREB phospho-S133 kit - 500 tests64CREPEG
-
CREB phospho-S133 kit - 10,000 tests64CREPEH
-
CREB phospho-S133 kit - 1 x 96 tests64CREPET
-
CREB phospho-S133 kit - 50,000 tests64CREPEY
-

Companion products

DescriptionCat. noProduct insertMSDS
CREB phospho-S133 kit control lysate64CRETDA
-
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF

Literature