Pcreb ELISA Kit
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- Target
- Pcreb
- Reactivity
- Human
- Detection Method
- Colorimetric
- Method Type
- DNA-Binding ELISA
- Application
- ELISA
- Purpose
- DNA-binding ELISA that facilitate the study of transcription factor activation in mammalien tissue and cell culture extracts.
- Brand
- TransAM®
- Sample Type
- Cell Extracts, Tissue Samples
- Analytical Method
- Quantitative
- Specificity
- TransAM pCREB Kits are tested for sensitivity in detecting phosphorylated CREB in nuclear extracts from WI-38 VA13 cells that are either unstimulated or stimulated with forskolin.
- Characteristics
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Transcription factors are DNA-binding proteins that tightly regulate gene expression. They consist of two distinct domains - one that displays high affinity for a specific DNA sequence and one that confers transcriptional activity. Transcription factors are activated by phosphorylation of specific residues or by processing bound inhibitory proteins. Understanding and quantifying transcription factors is essential for the study of cell functions in relation to differentiation, brain activity, immune response, inflammation and various disease states. TransAM® Kits are sensitive, non-radioactive transcription factor ELISA kits that facilitate the study of transcription factor activation in mammalian tissue and cell extracts.
TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. 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. - Components
- One or five 96-well plate(s) with plate sealer(s), primary antibody, HRP-conjugated secondary antibody, wild-type and mutated competitor oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing and Stop Solutions.
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- Application Notes
- Optimal working dilution should be determined by the investigator.
- Comment
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These extracts are diluted down to 0.625 μg/well and assayed using the TransAM pCREB Kit. The ratio of the signals from the stimulated over the unstimulated cells must be above 2. Lot No. 33614014 was developed for 2.5 minutes. It gave a ratio of 6.7 (Figure 1). The basal level of CREB expression, and this ratio, may vary depending on the cell type tested and the stimulation used. TransAM pCREB Kits are also tested for specificity in detecting CREB activation. TransAM pCREB assays are performed in the presence of an excess of oligonucleotide containing a wild-type or mutated CREB consensus binding site (Figure 2). At 20X excess, the wild-type oligonucleotide prevents CREB binding to the probe immobilized on the plate. Conversely, the mutated oligonucleotide has little effect on CREB binding.
- Assay Time
- 5 h
- Plate
- Pre-coated
- Restrictions
- For Research Use only
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- Storage
- 4 °C/-20 °C/-80 °C
- Storage Comment
- Except for the cell extract that must be kept at -80°C, the kit components can be stored at -20°C prior to first use. Then, we recommend storing the kit at 4°C except for the pCREB antibody, oligonucleotides, DTT, Protease Inhibitor Cocktail and Herring sperm DNA that should be kept at -20°C, and the cell extract at -80°C. This product is guaranteed for 6 months from date of receipt.
- Expiry Date
- 6 months
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- Target
- Pcreb
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