Technology

Standard methods for studying transcription factors and gene regulation include gelshifts, western blot and reporter gene assays, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. Marligen’s Chemiluminescent Transcription Factor Microplate Assays employ a proprietary assay technology and chemiluminescent detection that offers superior sensitivity, broad dynamic range and excellent reproducibility compared with traditional methods or with commercially available transcription factor ELISAs. The assays do not require the use of radioactivity, and results can be obtained in under four hours. The convenient stripwell format enables testing of from one to 96 samples in each plate, and the chemiluminescent signal is measured with standard 96-well plate luminometers. 

Marligen’s assays measure the binding of activated transcription factors to their cognate DNA binding sequences. First, nuclear extracts prepared from control or experimental cells or tissues are incubated with a biotin-labeled DNA probe containing the appropriate binding sequence and a single-stranded capture sequence subsequently used to immobilize the complex. A reagent is added to digest DNA probes that have not bound transcription factors, which dissociates the capture sequence from the biotin label unless the complex has been protected by the interaction of the DNA probe and activated transcription factors. Probes containing bound transcription factors are then captured onto the surface of the microplate, unbound label is washed away, and bound probes are detected using a streptavidin-alkaline phosphatase conjugate and a chemiluminescent substrate. The amount of signal generated is proportional to the amount of transcription factor in the nuclear extract that has bound to the labeled probe. A schematic of the assay is shown in Figure 1.

Figure 1: Summary of protocol for Marligen’s Chemiluminescent Transcription Factor NF-κB Microplate Assay.

Features and Benefits

• Detects Activation in Small Samples

Figure 2 : NFκB activation was observed in less than 0.1 μg of nuclear extract from TNF-α stimulated HeLa cells.

• Detects Multiple Transcription Factor Complexes

Figure 3: Marligen’s Chemiluminescent AP-1 Microplate Assay detects both c-jun homodimers and c-jun-c-fos heterdimers, obviating the need for specific antibodies to multiple protein forms or splice variants.

• Correlates with Marligen’s Multiplex Transcription Factor Assays. 

Figure 4A Figure 4B

Figure 4: Changes in the activity of multiple transcription factors are observed when 10 μg of nuclear extract from PMA treated cells are tested on Marligen’s 20-plex assay (Figure 4A). Single Analyte Chemiluminescent assays require less sample and can be used to further characterize individual responses, Figure 4B shows data from PPAR assay.