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HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
Shipping box for Revvity reagent kits
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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| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 16 µL |
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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Product Variants
Unit Size: 96 assay points
Part #:
64CREPET
List Price
USD 703.20
Your online price:
Unit Size: 500 assay points
Part #:
64CREPEG
List Price
USD 2,271.53
Your online price:
Unit Size: 10,000 assay points
Part #:
64CREPEH
List Price
USD 13,214.42
Your online price:
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
Shipping box for Revvity reagent kits
HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
Product information
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.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 1
Lysis Buffer 3
|
| Molecular Modification |
Phosphorylation
|
| Product Group |
Kit
|
| Sample Volume |
16 µL
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Metabolism/Diabetes
NASH/Fibrosis
Oncology & Inflammation
|
| Unit Size |
96 assay points
|
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HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
HTRF Human & Mouse Phospho-CREB (Ser133) Detection Kit, 96 Assay Points
How it works
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.
Assay validation
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.
Simplified pathway
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.
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