Figure 5. Binding affinity of C4 protein with dsDNA and ssDNA.

(A) Protein-DNA binding assay for C4 binding double strand BSCTV DNA. BSCTV fragments digested by EcoRI were end-labeled with [α- ³²P]dATP and Klenow polynucleotide kinase as a probe. Increasing amounts of C4 proteins (2–5 µg) were incubated with 50 ng probes at 22°C for 30 min. Gels were dried and the migration of labeled DNA was detected by a PhosphorImager. (B) Increasing amounts of C4 protein mixed with 0.5 µg 1 kb linear dsDNA ladder. DNAs were detected by visualization of ethidium bromide stained gels. C) Increasing amounts of C4 protein mixed with 0.2 µg M13 ssDNA. DNAs were detected by visualization of ethidium bromide stained gels. All mixtures were analyzed in 0.7% nondenaturing agarose gels in TBE buffer. The protein/DNA complexes are indicated.