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TransAM® GATA

dna-binding ELISA for activated GATA 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® GATA Transcription Factor ELISA Kits

TransAM GATA Family and GATA-4 Kits provide everything needed to study activated GATA binding protein, including a positive control extract. The GATA Family Kit measures GATA-1, GATA-2 and GATA-3 from human extracts, while GATA-4 can be detected separately using the TransAM GATA-4 Kit with human extracts. Click the GATA Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® GATA Family 2 x 96 rxns 48296 $1,125 Buy Now
TransAM® GATA-4 1 x 96 rxns 46496 $665 Buy Now
5 x 96 rxns 46996 $2,795 Buy Now

GATA Transcription Factor Info

The GATA binding protein family of transcription factors consists of six members, GATA 1-6, that bind to the (A/T)GATA(A/G) nucleotide motif.  GATA family members are grouped into two subfamilies based on structural and expression comparisons. GATA-1, -2 and -3 are expressed predominantly in hematopoietic cells, and regulate differentiation and gene expression in T-lymphocytes, erythroids and megakaryocytes. GATA-4, -5 and -6 are found mainly in the heart and gut, and are involved in regulation of cardiogenesis and gut development. GATA-1 is a transcriptional activator that is important for erythroid development; mutations in GATA-1 are associated with familial blood disorders. GATA-2 is a transcriptional activator that is expressed in hematopoietic progenitors and also in nonhematopoietic embryonic stem cells. GATA-3 is expressed mainly in T-cell lineage cells and in some nonhematopoietic cells, such as embryonic stem cells. GATA-4 is the transcriptional activator of several cardiac muscle-specific genes, as well as an activator of ANF.

 
 
 
Figure 1: Monitoring GATA family member activation using the TransAM GATA Family Kit.

GATA-1, -2 and -3 activation were assayed using the TransAM GATA Family Kit. 1:1000 dilutions of each antibody in the kit were tested using 5 µg/well of nuclear extract prepared from an unstimulated cell line: GATA-1 and -2 were tested with K-562 and GATA-3 with Jurkat. Assays were performed in the absence or presence of 20 pmol of competitor oligonucleotide that contains either a wild-type or mutated GATA consensus binding site. Note that the wild-type oligonucleotide reduces GATA binding by over 90%, while incubation with the mutant GATA competitor oligo has a limited effect on GATA-1, -2 and -3 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, two or five GATA 96-well plates with plate sealers, GATA-1, -2 and -3 or GATA-4 primary antibodies, HRP-conjugated secondary antibody, GATA wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffer, and Developing and Stop Solutions. 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.