Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes
Transcrição
Regulatory proteins, like transcription factors, recognize and bind to specific DNA sequences in promoter regions, regulating gene expression.
To detect specific transcription factor-DNA interactions using the electrophoretic mobility shift assay, begin with a tube containing a desired transcription factor and specific short, infrared, fluorescent dye-labeled DNA probes in a binding buffer. Incubate in the dark, preventing dye photobleaching.
The buffer's ionic strength and pH provide suitable conditions for the transcription factor to identify and bind specific binding sequences on the DNA probe. Magnesium ions in the buffer further stabilize the protein-DNA complexes.
Add a suitable loading dye to the mixture, helping visualize electrophoresis progression. Load this mixture and DNA probes into the wells of a precast, non-denaturing polyacrylamide gel.
Perform electrophoresis in the dark.
The applied electric field causes low molecular weight, unbound, negatively-charged DNA probes to move through the gel toward positively-charged anodes. However, the large protein-DNA probe complexes — with a higher molecular weight — migrate slowly, leading to a delayed migration and shift in the mobility of DNA in the gel.
Post-electrophoresis, scan the gel using an infrared imaging system, visualizing the infrared fluorescent dye-labeled DNA probes.
A free DNA probe band is at the bottom of the gel, while protein-DNA complexes appear as a shifted band on the gel, indicating protein-DNA complex formation.
