EMSA of GST-Cdc6. (A) Buffer only (lane 1)
and GST only (1000 ng, lane 2) were used as controls. Thirty nanograms
of the GST-Cdc6 fusion was used in lanes 3–8. One microgram of the
anti-Cdc6 antiserum (lane 4), 3 μg of rabbit preimmune serum (lane
5), and 0.2 μg of a monoclonal anti-GST antibody (lane 6) were
incubated with the GST-Cdc6 on ice for 60 min before the addition of 10
fmol of the 283-bp ARS1 probe. Lane 8 contains 1 mM ATP
in the reaction mixture. Lane 7 is identical to lane 3 and is a no-ATP
control. The arrow on the right indicates the
supershifts. (B) Buffer only (lanes 1, 5, 9, and 13) was used
as a control; 30, 60, and 90 ng of GST-Cdc6 were used with various DNA
probes: 180 bp of ARS1, 242 bp of the polylinker DNA
fragment from the plasmid pBlueScript, 283 bp of ARS1,
and 308 bp of HMR DNA.