Total CREB Transcription Factor Assay Kit (Colorimetric)
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Functional Studies - Total CREB Transcription Factor Assay Kit (Colorimetric) (AB207201)
Nuclear extracts from WI-38 cells (Gray) and WI-38 cells. Stimulated with Forskolin (Black) were assayed from 0.625 - 10µg/well for CREB activation using the Total CREB Transcription Factor Assay Kit.
Nuclear extracts from WI-38 cells (grey) and WI-38 cells stimulated with Forskolin (black) were tested for CREB activation. These results are provided for demonstration purposes only.
Product details
Total CREB Transcription Factor Assay Kit (Colorimetric) (ab207201) is a high throughput assay to quantify total CREB in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor binding.
A specific double stranded DNA sequence containing the total CREB consensus binding site (5’ –TGACGTCA– 3’) has been immobilized onto a 96-well plate. Total CREB present in the nuclear extract specifically binds to the oligonucleotide. Total CREB is detected by a primary antibody that recognizes an epitope of total CREB accessible only when the protein is bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects total CREB in human, mouse, rat and monkey samples.
Key performance and benefits:
- Assay time: 3.5 hours (cell extracts preparation not included).
- Detection limit: < 0.5 µg nuclear extract/well.
- Detection range: 0.3 – 10 µg nuclear extract/well.
CREB (Cyclic AMP Response Element-Binding protein) is a member of a large family of structurally related transcription factors that includes AFT1-4, c-Fos, c-Jun, c-Myc and C/EBP. The members of this family, named bZIP, share a dimerization domain with a leucine zipper motif and a DNA-binding domain rich in basic residues (lysines and arginines). CREB proteins specifically recognize the cAMP-responsive element promoter site.
Alternative splicing of the CREB gene yields several forms of CREB protein. The three most abundant forms are CREBα (341 aa), also called CREB1, CREBΔ (327 aa) and CREBβ (301 and 387 aa), which are present in human, rat and mouse tissues. Two other gene products highly homologous to CREB-1 have been characterized: activating transcription factor 1 (ATF-1) and cAMP response element modulator (CREM).
The CREB family members bind to the CRE promoter site as homo- and heterodimers. The ratio between these homo- and heterodimers, which depends on the cell type, regulates the CREB transcriptional activity as the homodimers have a longer half-life than CREB/ATF-1 heterodimers.
The CREB proteins activate transcription of target genes in response to a diverse array of stimuli, such as peptide hormones, growth factors and neuronal activity. Activation of CREB is mediated by a variety of protein kinases including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs) and Ca2+/calmodulin-dependent protein kinases (CaMKs) that phosphorylate CREB at the Ser-133 residue. Phosphorylation of Ser 133 is required for CREB-mediated transcription but not for dimerization and DNA-binding activity. Phosphorylation does, however, increase CREB's affinity for its promoter site. Upon cell stimulation with forskolin, an activator of adenylyl cyclase, the kinases are activated by cAMP production and translocate to the nucleus where they phosphorylate CREB at Ser 133. Therefore, CREB is almost exclusively nuclear in both unstimulated and stimulated cells. A cofactor, CREB-binding protein (CPB), specifically binds to phosphorylated CREB to enhance transcriptional activity.
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Biological function summary
CREB contributes significantly to cellular processes such as proliferation differentiation and survival through transcriptional regulation. CREB does not function alone; it frequently associates with other proteins forming complexes to exert its regulatory roles. Additionally CREB influences the development of memory and learning in neurons highlighting its extensive involvement in neurophysiological processes.
Pathways
CREB plays a substantial role in the cAMP signaling pathway as well as the mitogen-activated protein kinase (MAPK) pathway. Through its interaction with kinases and other signaling proteins CREB influences numerous downstream targets affecting cellular responses to external stimuli. Notably protein kinase A (PKA) activates CREB by phosphorylation facilitating its role in various signal transduction pathways and linking it to downstream transcriptional events.
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