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TransAM® NF-YA

dna-binding ELISA for activated NF-YA 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® NF-YA Transcription Factor ELISA Kit

TransAM NF-YA Kits provide everything needed to study Nuclear transcription Factor Y, alpha (NF-YA), including a positive control extract. The kit can be used with human, mouse and rat extracts. See the NF-Y Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® NF-YA 1 x 96 rxns 40396 $665 Buy Now
5 x 96 rxns 40896 $2,795 Buy Now

NF-Y Transcription Factor Info

Nuclear transcription factor Y, alpha (NF-YA), also known as CAAT-box DNA binding protein, subunit A (CBF-A) is a transcription factor that binds to the CCAAT motif of various genes, such as type 1 collagen, albumin and beta-actin. NF-Y is a ubiquitously expressed heterodimeric transcription factor composed of three subunits (NF-YA, NF-YB and NF-YC). All three subunits are required for constitution of a functional binding complex to the CCAAT box. Most of the sequence-specific interactions of the trimer are made by NF-YA, therefore, NF-YA represents the regulatory subunit of the NF-Y trimer. CCAAT boxes are found in the regulatory region of 30% of genes, including housekeeping genes as well as inducible and cell cycle-regulated genes. The role of a CCAAT box as a cis-acting promoter element depends on its ability to interact with NF-Y. Methods to measure overall levels and modifications of the NF-YA subunit are crucial to understanding transcriptional regulation of the numerous CCAAT box-containing genes.

 

 
 
Figure 1: Monitoring NF-YA activation with the TransAM NF-YA Kit.

Different amounts of nuclear extracts from Jurkat cells were tested for NF-YA activity by using the TransAM NF-YA Kit. Note that incubation with wild-type NF-Y competitor oligonucleotides reduces NF-YA binding by over 80%, while incubation with mutated NF-Y competitor oligos had no effect on NF-YA binding to DNA.

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

One or five NF-YA 96-well assay plate(s) with plate sealer(s), NF-YA primary antibody, HRP-conjugated secondary antibody, NF-Y wild-type and mutated oligonucleotides, positive control cell extract, Dithiothreitol, Protease Inhibitor Cocktail, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, Developing and Stop Solutions and Herring Sperm DNA. 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.