Protein binding sequences in the probes were mined from the TRANSFAC database and the literature to have the greatest affinity and specificity. The specificity of each binding site has been confirmed experimentally by a combination of methods including gel shifts, ELISAs, competition assays and Western blotting to verify the identity of the bound proteins. Results are reproducible, with CVs of <10% routinely achieved.

Technology

The technology utilizes a strategy similar to nuclease protection assays. A schematic diagram of the assay technology and workflow is shown in Figure 1.

Figure 1: Summary of protocol for Marligen’s Multiplex Transcription Factor Assay.

Marligen’s assays have been designed to facilitate a multiplex format, enabling detection and quantitation of many activated transcription factors simultaneously. Click here for more details about the use of Luminex xMAP® technology and instrumentation [goes to A5].

Currently, Marligen offers 20 different assays in multiplex format. These assays include:

AP-,1AP2, AR, CRE-ATF, CREB, EGR, E2F1-5, E2F6, GATA, HNF, HNF4, ISRE, Myc-Max, NF-1, NF-κB, PPAR, P53, SMAD2/3, SP1, YY1

Using this technology, up to 96 samples can be analyzed in less than 3 hours. Assays are performed by first incubating nuclear extracts with a mixture of labeled probes followed by the addition of a digestion reagent. Individual assays rely on the specific binding of transcription factors to cognate recognition sequences to protect labeled probes from digestion. Undigested probes are then captured onto the surface of fluorescently-labeled microspheres to provide a signal corresponding to the amount of activated transcription factor present within the test sample. Signals are detected with a compact flow cytometer as each microsphere passes through the Luminex® instrument.

Features and Benefits

Marligen’s revolutionary approach provides a wealth of biologically relevant data in a fraction of the time required for conventional gel shifts. The assays can be performed in less time than it takes to run standard ELISAs, while offering significantly more information per sample. This innovative technology offers unique benefits to researchers studying gene regulation.

Easily Identify Complex Responses Regulating Gene Expression

Unlike conventional methods such as EMSAs, each assay measures the presence of multiple transcription factors simultaneously, allowing powerful comparisons of different cell lines and treatments.

Figure 2. Multiplex assays measure the presence of multiple transcription factors simultaneously, allowing for powerful comparisons within cell and tissue models. Using 10 μg of nuclear extract, significantly different profiles of transcription factor activation are observed for K562 cells treated with Hemin (left panel) and with PMA plus ionomycin (right panel).

More Sensitive than ELISAs

The lower limit of detection is significantly better than those achieved with either ELISAs or EMSAs, which routinely require 10-50 ug of extract per analyte.

Figure 3. Different amounts of nuclear extracts isolated from PMA-treated and untreated THP-1 cells were assayed using Marligen’s Multiplex Transcription Factor Assay and a commercially available ELISA for detecting PPAR-γ.

Conserves Valuable Samples

The amount of sample required to detect and quantitate changes in the activity of a transcription factor compares very favorably with other methods such as EMSAs and ELISAs.

Figure 4. Comparison of the amount of extract necessary to obtain transcription factor binding data per binding site. The upper and lower ranges depend upon the cell types used, the basal levels of transcription factor activity and the levels of induction achieved upon treatment.

Powerful Tool for Identifying Effects of Drugs

Figure 5. Nuclear extracts isolated from untreated THP-1 cells and THP-1s treated with a proprietary drug were evaluated on a 28-plex. Data was plotted on a scatter plot to clearly distinguish differences between untreated and treated cells. Significant differences between the conditions are labeled.

Compatible With a Wide Range of Sample Types

Performance has been validated in many cell lines, primary cells and tissues from humans, monkeys and rodents including:

See our Application Notes for the Human Whole Blood and Tissue protocols.

An array of mouse tissues have also been used to make nuclear extracts including brain, liver, heart, lung, skeletal muscle, pancreas, kidney, spleen, bone marrow, and thymus. For a detailed protocol on how to prepare nuclear extracts from whole blood and selected tissues.