Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting.
A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes. The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A. After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation. Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase. The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt. A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator. The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment.
Granger-Schnarr
M
M
Institut de Biologie Moléculaire et Cellulaire, CNRS et ULP, Strasbourg, France.
Lloubes
R
R
de Murcia
G
G
Schnarr
M
M
D001426:Bacterial Proteins / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics; D009875:Operator Regions, Genetic; D011506:Proteins / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000302:isolation & purification; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004247:DNA; D004269:DNA, Bacterial; D011506:Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein