The set of transcription factors that include hepatocyte nuclear factor 1-alpha (HNF-1-alpha), HNF-1-beta, HNF-3-alpha, -beta, and -gamma, HNF4, and HNF6 is preferentially expressed in hepatocytes and plays an important role in liver-specific transcription. HNF-1-alpha was first identified as a DNA-binding protein capable of interacting with the promoters of several hepatic genes, including albumin, alpha1-antitrypsin, and beta-fibrinogen. HNF-1-alpha is composed of three domains, an amino-terminal dimerization domain, a DNA-binding domain belonging to the homeobox family, and a carboxyl-terminal domain that is essential for transactivation of target promoters. HNF-1-alpha and HNF-1-beta share strong homologies in both the dimerization domain and the DNA-binding domain which enable the two proteins to form heterodimers and bind to the same DNA sequences. However, HNF-1-alpha is stronger as a transactivator than is HNF-1-beta. HNF-1-alpha is expressed in polarized epithelia of different organs, including the liver, digestive tract, pancreas, kidney, spleen, thymus, testis, keratinocytes and melanocytes in human skin. HNF-1-alpha is crucial in controlling the postnatal functions of the liver, pancreas, and kidney and involving in various metabolic pathways of other organs through the transcriptional activation of genes that play key roles in phenylalanine catabolism, pancreatic beta-cell glucose-sensing, and renal proximal tubular reabsorption of glucose and several other metabolites. In addition, HNF-1-alpha regulates the expression of acute phase proteins, such as fibrinogen, c-reactive protein, and interleukin 1 receptor, which are involved with inflammation. Moreover, HNF-1-alpha might play a possible tumor suppressor role.
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
Other Materials Required
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation <lIκBenchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Transcription factor assay of HNF-1-alpha from nuclear extracts of HepG2 cells or HepG2 cells treated with IL-6 with RayBio HNF-1-alpha TF Activity Assay Kit.
Transcription factor assay of HNF-1-alpha from nuclear extracts of HepG2 cells or HepG2 cells treated with IL-6 with the specific competitor or non-specific competitor. The result shows specific binding of HNF-1-alpha to the HNF-1-alpha DNA binding site.
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.