DETECTION OF DNA BINDING PROTEINS BY GEL RETARDATION

PURPOSE: To determine whether a nuclear extract contains a protein that binds specifically to a given DNA fragment.
 

PROTOCOL:
 

a) End-label DNA fragment of interest

b) Incubate fragment with soluble extract from nuclei

c) Load mixture on acrylamide gel and electrophorese. In some cases, the binding between DNA and protein may be so fragile that the mixture is loaded while the current is on. Delays between loading the first sample and the last sample may result in a 'smiling' appearance to the gel.

d) Autoradiograph gel.

a) Pre-treatment of extract with heat (90°C, 5min.) or proteinase K may be used to demonstrate that the factor binding the DNA is a protein, and not RNA.

b) Unlabeled competitor:

Non-specific (eg. poly-dI:dC, E.coli DNA). If addition of non-specific competitor eliminates a retarded band, then that band is due to binding by a protein that recognizes DNA in general, and not specific sequences of DNA.

Specific - A purified, unlabeled DNA fragment can eliminate a retarded band when added to the extract, while other DNA fragments can not, then binding is said to be specific. It is often a good idea to do a control in which an excess of unlabeled probe DNA competes with the labeled probe.

When no nuclear extract is added, no binding occurs. In the presence of extract some of the DNA is complexed with protein, and moves more slowly through the gel. When extract and an excess on unlabeled competitior is added, most of the labeled DNA remains unbound.