Fig. 4.

Dot blot assay for ATP binding. GcsT in buffer comprised of 50 mM Tris-HCl pH 8.5, 50 mM NaCl, 3 mM β-me and 4 mM MgCl₂ was immobilized on a nitrocellulose membrane and incubated with labeled 50 μM of γ⁻³²P ATP. (A) Nitrocellulose dot blot autoradiograph on X-ray film, showing effect of increasing concentration of protein, ATP and EDTA on ATP binding. (B) Intensity of spots representing bound radiolabelled ATP was plotted against increasing EDTA concentration. Bar diagram representing decrease in ATP binding with increasing EDTA concentration. (C) Bar diagram showing increase in ATP binding with increasing protein concentration. (D) Bar diagram showing displacement of radiolabeled ATP by unlabelled ATP as revealed by decrease in spot intensity.