EMSA (Electrophoretic Mobility Shift Assay) kits are used for monitoring transcription factor - DNA binding activities and activation. Upon activation, the TF can recognize and interact with the unique DNA binding sequence and form a protein/DNA complex, which can be separated from free DNA due to the differences of their electrophoretic mobility in native (non-denaturing) polyacrylamide gels.
100+ non-radioactive EMSA Kits are available. The probes are pre-labeled with biotin, and all reagents needed are included in the kit.
Please note: The Nuclear Extraction Kit, Cat# SK-0001-SO needs to be purchased separately.
In the TF filter plate assays, a specific biotin-labeled TF DNA binding sequence is mixed with nuclear extracts to allow formation of TF-DNA complexes. A filter plate is used to retain bound DNA probe and remove free probe. The bound pre-labeled DNA probe is then eluted from the filter and collected for quantitative analysis through DNA plate hybridization. The captured DNA probe is further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) using a microplate luminometer. This "EMSA on a plate" assay allows you to monitor the activation of one TF in vitro in up to 96 samples at the same time.
Please note: The Nuclear Extraction Kit, Cat# SK-0001-SO needs to be purchased separately.
TF activation profiling plate arrays are used for monitoring the activities of multiple TFs simultaneously. A series of biotin-labeled probes are made based on the consensus sequences of TF DNA-binding sites. When the probe mixtures are incubated with nuclear extracts, individual probes will find their corresponding TFs and form TF/probe complexes, which can be easily separated from free probes through a simple spin column purification. The bound probes are then detached from the complexes and analyzed through hybridization to a plate, in which each well is specifically pre-coated with complementary sequences of the probes.
To characterize transcription factors (TFs) that bind to a specific promoter or regulate the expression of a specific gene via its upstream promoter, two common approaches are applied. First is to employ gel shift assays with DNA binding sites of TFs that are silico-identified within the promoter. Second is the removal or knockout of the binding site(s) of a specific TF in order to measure whether the expression of a promoter-linked reporter is increased or decreased. Often, a series of reporter constructs with the promoter deletions or mutations is required because many binding sites of one or a few TFs are present within a promoter. Promoter Binding TF Profiling Plate Arrays represent a fast method to facilitate the characterization of promoters through a revised TF activation array. This assay will help to test whether selected 48 or 96 TFs bind to the promoter or not.
TF ELISA kits are highly sensitive and specific assays with a simple and optimized procedure. The 96-well (8X12 strip) clear plate is pre-immobilized with the TF consensus sequencing oligo. The activated TF in the nuclear extract or the whole cell lysate is added to the well and binds to the oligo. The activated TF is detected with a specific antibody against the TF subunit and a HRP-conjugated secondary antibody. The assay utilizes colorimetric detection, which can be easily measured by spectrophotometry.
More ELISA-like transcription factor activity assays can be found here