BIOL 200 Lecture Notes - Lecture 6: Dna-Binding Domain, Sv40, Deoxyribonuclease I
27 May 2016
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Document Summary
Antibodies, substrates: beads have affinity for specific types of proteins. Identifying dna-binding protein sequences: dnase i footprinting. Regions on a dna bound by proteins are protected from digestion by enzymes. These strands appear as a gap when run thru gel electrophoresis and compared to non- protein-bound dna strands: emsa (electrophoretic mobility shift assay) Dna sequences bound to proteins will move through gel electrophoresis at a slower rate. Does not provide specific dna sequence to which protein binds. Identify binding location of a protein (sequence unknown) using dnase i footprinting. Copy the specific dna sequence that this tf binds to. Run under emsa and find which fractions contain the tf in question. Sp1 transcription factor tested on adenovirus dna (positive control) and sv40 dna. Plasmid 1 (contains gene-encoding certain transcription factor) + plasmid 2 (contains reporter gene; must be a gene not naturally expressed in cell) Change observed by the presence of tf in plasmid 1 into cell.
