Technical Approaches toward identifying Proteins with DNA binding activities
An important class of protein-protein interactions is that of proteins interacting with specific nucleic acid sequences, such as in the promoter elements of genes. These interactions involve not only interactions between several proteins associated with transcription and repair, but also critical interactions with defined nucleotide sequences.
1. Electrophoretic Mobility Shift Assay (EMSA): This technique has been instrumental in identifying binding sites for . Here, a radioactively labeled promoter or enhancer sequence is incubated with a nuclear extract containing a DNA binding protein. That DNA-protein complex is run on one lane of a gel and the DNA alone is run on another lane. The DNA-protein complex lane will have an extra slower running band (corresponding to the DNA-protein complex) in addition to the faster running free DNA band.
The DNA-binding proteins seen in EMSA can be identified using 2-D electrophoresis coupled with mass spectrometry. T
2. Chromatin immunoprecipitation (ChIP) is the best test to insure your protein binds with your promoter. In this assay you fix and lyse your cells, shear the DNA and then use an antibody which binds to your promoter to precipitate your DNA. For example, you might use the ChIP assay to assess histone H4 acetylation at theIl4 and IFNg regulatory regions. To do this you could purify T cells from the spleen and lympho nodes of mice. Chromatin complexes are then immunoprecipitated with antiboides to acetylated histones H4(+). PCR primers specific for the Il4 promoter is then used to amplify the precipitated DNA.