c-Fos EZ-TFA Trancription Factor Assay Colorimetric Kit

c-Fos EZ-TFA Trancription Factor Assay Colorimetric Kit

Upstate (Millipore)

1 plate

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 colorimetric detection that can be read in a spectrophotometric plate reader. 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.

Activity Assay

• Human
• Mouse
• Rat

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 Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death.

FUNCTION: SwissProt: P01100 # Nuclear phosphoprotein which forms a tight but non- covalently linked complex with the JUN/AP-1 transcription factor. In the heterodimer, c-fos and JUN/AP-1 basic regions each seems to interact with symmetrical DNA half sites. Has a critical function in regulating the development of cells destined to form and maintain the skeleton. It is thought to have an important role in signal transduction, cell proliferation and differentiation.
SIZE: 380 amino acids; 40695 Da
SUBUNIT: Heterodimer with JUN. Interacts with DSIPI; this interaction inhibits the binding of active AP1 to its target DNA. Interacts with MAFB (By similarity).
SUBCELLULAR LOCATION: Nucleus.
SIMILARITY: SwissProt: P01100 ## Belongs to the bZIP family. Fos subfamily. & Contains 1 bZIP domain.

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.

• Rabbit anti-c-Fos (Part No. 90557): (70-545 and 70-546) One vial containing 30 μL Rabbit anti-c-Fos.
• Mouse anti-c-Jun (Part No. 2003735): (70-540 and 70-546) One vial containing 15 μL Mouse anti-c-Jun monoclonal antibody.
• 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.
• HeLa Nuclear Extract, 4 hr Serum Response Treatment (Part No. 90558): One vial containing 25 μL of 10% serum treated HeLa nuclear extract. This extract is provided as a positive control for 70-545 and 70-546 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.
• 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 70-540 and 70-546 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, Colorimetric) Catalog #70-500: One universal kit containing 80 mL TFA Buffer, one streptavidin-coated stripwell plate, 12 grams of blocking reagent, 250 μl 10X annealing buffer, 12 mL TMB/E, 12 mL stop solution, 30 μl Gt X Rb HRP secondary antibody, 30 μl Gt X Ms HRP secondary antibody. A detailed manual is also included with the 70-500 Universal TFA component. The procedures described in the manual should be consulted as well as the specific product information in this 70-540/545/546 product specific manual.

Upstate

P01100

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

SGT545

Fos is a component of the AP-1 transcription factor (heterodimer with Jun, or Jun homodimer). Whereas Jun is expressed constitutively but activated following phosphorylation, the Fos promoter contains a serum-response element, and Fos is expressed following mitogenic stimulation, with immediate-early kinetics. AP-1 activity thus differs based upon whether or not the cells are activated to express Fos.

• FOS
• G0S7
• c-fos

c-Fos EZ-TFA Trancription Factor Assay Colorimetric Kit

NM_005252.2

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)