BD Biosciences' Mercury TransFactor profiling kit is a quick and convenient ELISA based system for studying DNA binding proteins. The system is comprised of a 96 well plate that is coated with oligos that contain DNA binding sequences for a particular transcription factor of interest. Cells are treated as desired and nuclear extracts are prepared. Extracts are incubated in wells that are coated with transcription factor specific DNA binding sequences. Mutant sequences are also provided as negative controls, as well as positive nuclear lysate. Detection of bound proteins is then achieved by a primary antibody that is subsequently detected by a secondary antibody conjugated to horse radish peroxidase. The bound immunocomplex is then detected with an HRP susbtrate reaction, and the absorbance of the substrate is read in a standard plate capable spectrophotometer.
The system is quick and easy compared to traditional detection of nuclear translocated proteins, circumventing time consuming microscopy techniques or traditional EMSAs that require radioactivity. Assuming that the nuclear extraction procedure is optimized for the cell system of use, it saves considerable time over microscopy and even western blot analysis of nuclear extracts. The kits are configured for the detection of various popular transcription factors including: HIF-1alphabeta, PPAR alpha, beta & gamma, oncogenesis kit 1 (DP-1, E2F-1, RB, p107, E2F-2, Sp-1), oncogenesis kit 2 (c-Myb, c-Myc, Max, USF1, USF2, p53), oncogenesis kit 3 (JIF-1alpha, HIF-1b, Egr-1, c/EBP, Oct1, Oct2), inflammation kit 1 (p50, p65, c-Rel, ATF2, CREB, c-fos), and inflammation kit 2 (c-jun, c-fos, FosB, JunD, Sp-1, Stat1). These systems are all 96 well format, so they are a fast and convenient system for detecting either 1 or multiple transcription factors that have translocated to the nucleus. The system is well suited for a large number of samples. The kits contain the necessary plate with specific oligos that contain DNA binding consensus sequences, mutant control oligos, extraction buffers, secondary and primary antibodies, TMB substrate, and detailed instructions. The assay itself takes about 3-4 hours.
The advantage of these kits is that not only can they analyze multiple transcription factors, but they are more sensitive than traditional gel-shift assays. They are designed for high throughput screening of many samples, something not easily achieved by standard techniques. Also, the addition of control and mutant oligos for detection are convenient for an internal calibration of the assay.
The only drawback to the system is in the nuclear extraction buffer. Anybody who has done nuclear extraction from mammalian cells knows that it sometimes takes optimization of the extraction buffers for the extracts to be purely nuclear. For such considerations it would be ideal to have capture antibodies to solely cytosolic and nuclear proteins in the wells as controls for the extraction procedure.
Overall, the BD Mercury TransFactor profiling kits are easy to use, convenient and highly specific for the detection of nuclear factors that have translocated to the nucleus. The 96-well based ELISA design is optimal for large sample profiling of even screening efforts. They also have an advantage of standard EMSAs in that they don’t require radioactivity.
Omar Perez, Ph.D.
Center for Clinical Science Research
Stanford University