The Non-Radioactive Jun/c-Fos Transcription Factor Assay kit is provided in a 96-well format. During the assay, the Capture Probe, a double stranded biotinylated oligonucleotide containing the flanked DNA binding consensus sequence for c-fos (5'- TGA(C/G)TCA -3'), is mixed with cellular (nuclear) extract in the Transcription Factor Assay Buffer provided. When incubated together, the active form of Jun and Fos contained in the nuclear extract binds to the consensus sequence. The extract/probe/buffer mixture is then directly transferred to the streptavidin coated plate. The active Jun/Fos transcription factor proteins is immobilized on the biotinylated double stranded oligonucleotide capture probe bound to the streptavidin plate well, and any unbound material is washed away. The bound c-Jun/c-Fos transcription factors are detected with specific primary antibodies, a mouse monoclonal anti-c-Jun and a rabbit anti-c-Fos. A highly sensitive HRP-conjugated secondary antibody is then used for detection. This provides sensitive chemiluminescent detection that can be read in a microplate luminometer or by a CCD camera-coupled imaging system. Included in the kit are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide. For Research Use Only; Not for use in diagnostic procedures
Activator Protein-1 (AP-1) is an inducible multimeric transcription factor complex associated with growth factor response, cellular proliferation, differentiation, apoptosis and stress response. The AP-1 complex is often composed of homodimers of the jun family of basic leucine zipper (bZip) proteins, or as heterodimers of jun family members and the related fos bZip proteins. The activity of both Fos (c-Fos, FosB, fra-1, fra-2) and Jun (c-Jun, JunB, JunD) family members are regulated on multiple levels including transcription, mRNA stability, post-translational modification by various MAP kinases, and protein turnover. Importantly, phosphorylation of these proteins is required for their transcriptional activity, often facilitating interaction with transcriptional co-activators. Once activated by stimuli such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or serum, phosphorylated jun:jun or fos:jun dimers bind to the palindromic AP-1 TPA response element (TRE) (TGAGTCA) and activate transcription of genes such as cyclin D1. Both Fos and Jun are also capable of interacting with other bZip factors of the CREB/ATF family, which recognize a distinct response element, the cAMP Response Element or CRE (TGACGTCA). The ability of members of each of these families to receive signaling inputs from distinct MAP kinases and direct transcriptional programs in a homo or heterodimer specific fashion contributes to the overall broad range of effects of AP-1 activity on cellular behavior.
c-Fos was originally identified as the cellular homolog of an oncogene (v-fos) encoded by the FBJ murine osteosarcoma virus. It is an obligate heterodimer of the Jun family, and its expression is rapidly induced upon exposure of cells to growth factors, cytokines, and various other intra- and extracellular signals. It harbors two distinct transactivation domains, at least one of which is phosphorylated, and gene knockout experiments demonstrate that c-fos is required for osteoclast development.
Jun family proteins contain a single activation domain regulated to a large degree by the JNK family of MAP kinases. Following activation, JNK associates with and phosphorylates c-Jun on serines 63 and 73 resulting in an increase in its ability to interact with other proteins including the CBP/p300 family of coactivators thus resulting in transcription activation.
- Affinity Binding Assay
- ELISA
- Activity Assay
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
The reagents in this kit have been optimized to reduce background signal while maintaining a high signal to noise ratio. Quality control testing is performed using the enclosed kit components while following the protocol described herein.
- Mouse anti-c-Jun (Part No. 2003735): (70-640 and 70-646) One vial containing 15 μL Mouse anti-c-Jun monoclonal antibody. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer (see 70-600 Universal Transcription Factor Assay product manual).
- AP-1 Capture Probe (Part No. 90555): One vial containing 220 μL biotinylated double stranded oligonucleotide probe (1 pmol/μL) containing the wild-type TRE consensus sequence (5'-TGA(C/G)TCA-3') for Jun/Fos.
- AP-1 Specific Competitor Oligonucleotide (Part No. 90556): One vial containing 20 μL (10 pmol/μL) specific competitor oligonucleotide. The AP-1 Competitor Oligonucleotide has the same TRE consensus sequence (5'-TGA(C/G)TCA-3') as the AP-1 Capture Probe but is not biotinylated and will not bind to the plate. It is provided as a specific competitor for transcription factor binding and can be used to demonstrate specificity. This control does not need to be run with each assay performed.
- TFA Negative Control Probe (Part No. 90271): One vial containing 20 μL biotinylated double stranded oligonucleotide probe (2 pmol/μL). Provided as an internal negative control, this non-specific competitor oligonucleotide does not contain the TRE Jun/Fos consensus.
- WI-38 Nuclear Extract (Part No. 2003764): One vial containing 25 μL of untreated WI-38 nuclear extract. This extract is provided as a positive control for 70-640 and 70-646 and is not intended for extract-to-extract, nor plate-to-plate comparisons. When using this control, you will notice a decrease in signal with repeated freeze/thaw cycles. This component must be stored at -80°C.
- EZ-TFA (Universal Transcription Factor Assay, Chemiluminescent ) Catalog #70-600: One universal kit containing 80 mL TFA Buffer, one streptavidin-coated stripwell plate, 12 grams of blocking reagent, 250 μl 10X annealing buffer, 4 mL chemiluminescent detection reagent, 8 mL chemiluminescent reaction buffer, 30 μl Gt X Rb HRP secondary antibody, 30 μl Gt X Ms HRP secondary antibody. A detailed manual is also included with the 70-600 Universal TFA component.
The Transcription Factor Assay is a triple temperature storage assay kit. Store kit components at the temperatures indicated on the labels and this insert until their expiration date.
• Store HeLa Whole Cell Extract at -80°C. Avoid repeated freeze-thaw cycles. Stable at -80°C for 6 months.
• Store the AP-1 Capture Probe, Competitor Oligonucleotides, Negative Control Probe, and all antibodies at -20°C.
• Store the Transcription Factor Assay (TFA) Buffer at -20°C. Avoid freeze-thawing of the TFA Buffer. Buffer can be quickly thawed in warm water prior to use. Buffer can be aliquotted and frozen.
• Store the TFA 96-well plate and the TMB/E and stop solutions at 4°C.
96 wells
- STAT3
- APRF
- FLJ20882
- MGC16063
1. Nuclear Extraction Kit (Catalog No. 2900) or components
2. Pipettor (both single and multi-channel)
3. Pipette tips
4. Distilled water
5. Microplate reader (wavelength 450nm/650nm)
6. Rotator or Plate shaker (optional)
7. Plate cover (optional)
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详细描述见链接:http://www.millipore.com/catalogue/item/70-640