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Marligen's Multiplex Transcription Factor Technology Marligen’s Multiplex Transcription Factor Assays employ a powerful multiplexing approach for simultaneously measuring the binding of many different transcription factors to their unique regulatory DNA elements. The technology dramatically accelerates studies ranging from basic research in signal transduction and proteomics to target validation, compound screening, toxicity testing and disease characterization. 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 multiplex 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. Currently, up to 50 different transcription factors can be measured simultaneously with Marligen’s multiplex Transcription Factor Testing Services. These assays include: AP-1, AP2, AML-1, AP4, AR, CRE-ATF, C/EBPα, C/EBPβ, C/EBPγ, CREB, E2F1-5, E2F6, EGR, ER, ETS, GATA, HFH2/3, HIC-1, HIF, HNF-1, HNF-3, HNF-4, HSF-1, ISRE, MEF-2, MTF1, c-Myb, Myc-Max, NFAT, NF-1, NFκB, NFY1, Octamer, p53, PAX6, PBX, PLZF, PPAR, SAF-1, SOX9, STAT, SREBP, SMAD1/5, SMAD 2/3, SP1, SRE, TGIF, TAL1, TCF/LEF and YY1 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. 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 μg 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 quantitative changes in the activity of a transcription factor compares very favourably 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 labelled. Compatible with a Wide Range of Sample Types
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. Please click here for a description of other products and services offered by Marligen. |
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