Non-Radioactive EZ-TFA™

Transcription Factor Assays

Detect transcription factor DNA binding activity in a convenient 96-well format
Adaptable for manual or high-throughput screening
Faster and more sensitive than radioactive gel-shift assays
Assay results in about 4 hours

Assay Overview

The Upstate EZ-TFA non-radioactive transcription assays from Millipore provide a fast, sensitive, and flexible method for detecting the DNA binding activity of transcription factors in whole cell or nuclear extracts in a convenient 96-well format in your choice of the colorimetric or the ultra-sensitive chemiluminescent format. These assays elegantly combine the principles of the electrophoretic motility shift assay (EMSA) and gel super-shift assays with the 96-well based enzyme-linked immunosorbent assay (ELISA), enabling manual or high throughput sample analysis with greater sensitivity than conventional EMSA assays. The entire assay takes less than 4 hours to complete with minimal hands-on time. The versatile set up allows for a flexible assay design and because the binding reaction occurs in solution, various parameters can be optimized such as probe titration, competitive oligonucleotide concentration, or treatment conditions.

Kit Components

The Upstate Grand EZ-TFA assays from Millipore are offered in a flexible format to suit the needs of the user. The kits are offered in both a colorimetric and a chemiluminescent version for both the Universal EZ-TFA kits as well as the target specific assays (please see the list below of extensive listing of more than 20 target specific kits available).

EZ-TFA Universal Kits

Include the 96-well streptavidin-coated plate, 5X TFA buffer, blocking reagent, secondary antibodies (mouse and rabbit), necessary protocols for the assay, and either colorimetric or chemiluminescent detection reagents depending on format of kit purchased.

Target Specific Kits

Include the EZ-TFA Universal kit module and the necessary components needed to look at the activity of the particular target in 96 wells (192 for family kits). This includes a highly specific primary antibody, biotinylated capture probes, competitor probes (non-biotinylated probes containing the DNA binding consensus sequence), and biotinylated TFA negative control probes (provided as background control and eliminates interference of nonspecific DNA binding). This allows for the end user to accurately test the DNA binding activity of the target, while allowing the option to also test other non-canonical transcription factor DNA binding consensus sequences.

In Millipore’s assay, the binding reaction is performed in solution and the DNA bound complex is then captured on a streptavidin coated plate, while in competitors’ assays the probe is pre-immobilized on the plate. Our format delivers increased flexibility when compared with similar ELISA based transcription factor assays.

The figure compares the EZ-TFA NFêB p65 Transcription Factor Assay (Millipore cat. no. 70-520) with a competitor’s assay. In this example, untreated and TNF-á treated HeLa cell nuclear extracts are incubated with an oligonucleotide containing the consensus DNA binding site for NFkB as the capture probe. Active NFêB in the sample binds to the probe and is detected with a specific primary antibody, followed by an HRP-conjugated secondary antibody and a colorimetric substrate reaction, which can be read on a standard microplate reader.

Find Primary Antibodies

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