Gene Cloning

342 Gene Cloning
(a)
F DP DPA
DNA-transcription
factor-antibody complex
DNA-transcription factor
complex
Free DNA
(b)
Transcription
factor binds here
Protein
Full length
1 Bound
2
3
4
Free
FL 1 2 3 4
– + – + – + – + – +
Bound
Free
Figure 11.15 The electromobility shift assay (EMSA). a) A labeled (*)
DNA fragment migrates according to its size during gel electrophoresis (lane F).
When bound by a protein the DNA fragment’s mobility during electophoresis is
reduced and therefore protein binding to DNA can be detected as a shift in
mobility (lane DP). Increasing the mass of the complex further by binding of a
second protein, in this case an antibody, results in another shift, often called a
supershift. Binding of the antibody to the complex is used to help confirm the
identity of the protein present in the original complex. b) The full length
fragment binds the transcription factor (lane FL). Four shorter overlapping
fragments are used to map the position of the transcription factor binding site.
The transcription factor binds to fragments 1 and 2, but not 3 and 4. The binding
site on the DNA must be within the common sequence between fragments 1
and 2.
compared with the free DNA. When visualized the position of the DNA–
protein complex will have shifted compared with the free DNA hence the
term electromobility shift assay. The assay normally requires the formation
of the DNA–protein complex in vitro with a radiolabeled DNA fragment.