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TransAM® HNF

dna-binding ELISAs for activated HNF transcription factors

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® HNF Transcription Factor ELISA Kits

TransAM HNF Family and HNF-1 Kits provide everything needed to study activated Hepatocyte Nuclear Factors (HNFs), including a positive control extract. The HNF Family Kit measures HNF-1, HNF-3α, HNF-3β and HNF-4α from human, mouse and rat extracts. HNF-1 can be detected separately using the TransAM HNF-1 Kit with human, mouse and rat extracts. Click the HNF Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® HNF Family 2 x 96 rxns 46296 $1,125 Buy Now
TransAM® HNF-1 1 x 96 rxns 46196 $665 Buy Now
5 x 96 rxns 46696 $2,795 Buy Now

HNF Transcription Factor Info

Hepatocyte Nuclear Factors (HNFs) are primarily required for cellular differentiation and metabolism. Unlike most transcription factor families, this group of evolutionarily conserved proteins does not have a common consensus-binding site. This makes assaying HNF family activity using classical EMSA or reporter assays cumbersome and time consuming. The TransAM HNF Family Kit contains multiple binding sites in each well, making it quick and easy to monitor HNF-1, -3α, -3β and -4α activity in a single assay. In addition, a TransAM HNF-1 Kit is available separately for researchers who want to monitor this important regulator of liver function.

Hepatocyte nuclear factor 1 alpha (HNF-1) is required for the expression of several liver-specific genes. It recognizes the consensus sequence 5'-GTTAATNATTAAC-3' and forms heterodimers with HNF-1 beta. Defects in HNF-1α are associated with maturity onset diabetes of the young type 3 (MODY3). HNF-3 α, β and γ are found in the liver, pancreas, intestine and lung. HNF-3 target genes include albumin, Apo B, transferrin, insulin-like growth factor binding protein 1, aldolase B and complement protein C. Hepatocyte nuclear factor 4 alpha (HNF-4) binds DNA as a homodimer to regulate the expression of HNF-1α, and may be essential for development of liver, kidney and intestine. Mutations in HNF-4 can lead to non-insulin dependent diabetes mellitus type 1.

 

 
 
Figure 1: Monitoring HNF-1 activity with the TransAM HNF-1 Kit.

Different amounts of nuclear extracts from untreated Hep G2 and HeLa cells are tested for HNF-1 activity using the TransAM HNF-1 Kit. Info

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 HNF-1 96-well plate(s) or two HNF Family 96-well plates with plate sealer(s), HNF-1 or HNF-1, HNF-3α, HNF-3β and HNF-4α primary antibodies, HRP-conjugated secondary antibody, HNF-1 or HNF family wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, 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.