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dna-binding ELISAs for activated CREB and phosphorylated CREB transcription factor

TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.

TransAM® CREB Transcription Factor ELISA Kits

TransAM CREB and TransAM pCREB Kits provide everything needed to study activated Cyclic AMP Response Element Binding (CREB), including a positive control extract. The TransAM CREB kit will detect total CREB, independent of phosphorylation state, in human, mouse, rat and monkey extracts, while the TransAM pCREB kit will detect CREB phosphorylated at Ser133 in human, mouse, rat and hamster extracts as well as phosphorylated CREM-1 and ATF-1. Click the CREB Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

Name Format Cat No. Price  
TransAM® CREB 1 x 96 rxns 42096 $665 Buy Now
5 x 96 rxns 42596 $2,795 Buy Now
TransAM® pCREB 1 x 96 rxns 43096 $665 Buy Now
5 x 96 rxns 43596 $2,795 Buy Now

CREB Transcription Factor Info 

Cyclic AMP Response Element-Binding (CREB) protein is critical in a variety of cellular processes such as cell proliferation, differentiation and hormonal control of metabolic pathways. CREB is a member of a large family of structurally related transcription factors and binds as a homodimer to specific DNA sequences called the cAMP-response element (CRE) (5´-TGACGTCA-3´). CREB proteins activate the transcription of target genes in response to a diverse array of stimuli, such as the introduction of peptide hormones or growth factors, and neuronal activity. CREB activation is mediated by a variety of protein kinases, including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs), and Ca+2 calmodulin-dependent protein kinases (CaMKs). These phosphorylate CREB at the Ser133 residue. Both phosphorylation at Ser133 and the binding of coactivator CBP (CREB-binding protein) are required to induce CREB-mediated transcription. Phosphorylation of CREB at Ser133 may play a role in the regulation of circadian rhythmicity. TransAM CREB and pCREB Kits contain antibody directed against CREB and phosphorylated CREB, respectively.

Figure 1: Measurement of phosphorylated CREB.

Human fibroblast WI-38 cells are stimulated with Forskolin for 30 minutes. Increasing amounts of nuclear cell extracts are assayed using the TransAM pCREB Kit.

The TransAM® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

Why use TransAM® transcription factor ELISAs?

  • Up to 100-fold more sensitive than gelshift assays
  • Eliminates the use of radioactivity and the need to run gels
  • Results in less than five hours
  • Colorimetric readout enables easy, quantitative analysis with spectrophotometry at 450 nm
  • 96-stripwell format enables both high and low throughput

How TransAM® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

Contents & Storage

Each TransAM CREB or pCREB Kit contains CREB or pCREB primary antibody, HRP-conjugated secondary antibody, CREB wild-type and mutated oligonucleotides, WI-38 nuclear extract (positive control), Dithiothreitol, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis Buffer, 10X Washing Buffer, 10X Antibody Binding Buffer, Developing Solution, Stop Solution and one or five CREB 96-well assay plate(s) with plate sealer(s). Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.