Liquid scintillation counting, binding

The Importance of divalent Ions shows up most clearly In quantitative binding experiments using C-labeled pyran copolymer. In these experiments, whole ei throcytes - not Isolated membranes - were Incubated at room temperature for 10 minutes with the desired concentration of pyran copolymer In buffer. The cells were then sedimented by centrifugation, washed twice with fresh buffer, disrupted with detergent, and the radioactivity detennined by liquid scintillation counting. 

Fig. 2. BA-binding activity of the soluble CCKBP. Isolated chloroplasts were suspended in 2 M NaCl with 0.05 M Tris-HCL buffer (pH 7.6). The suspension was incubated in an ice bath for 15 min followed by centrifugation at 23 000 Xg for 10 min. The supernatant was desalted with Sephadex G-25. The proteins eluted by Tris buffer were subjected to (NHJ..SO4 fractionation. The pellets were suspended in Tris buffer and then dialyzed against Tris buffer at 4°C for 4 h. Usually a 1 ml sample (ca. 500 jug protein) was added to an Eppendorf tube containing 0.12 /iCi p H]-BA. After 30 min at 4°C the mixtures were dialyzed for 8 h (at 4°C) against Tris buffer containing 10 mM NaCl and 0.5% charcoal powder. The radioactivity in each sample was counted with 0.2 ml dialyzed protein solution, plus 7 ml of liquid scintillation solution...

To 5 ml polystyrene tubes were added successively 0.1 ml tritiated tracer (about 7,500 dpm), 0.1 ml PG standard or biological extract and 0.1 ml of a dilution of the antiserum such that the initial binding in the absence of standard or unknown PG is 40-50% of the total radioactivity. All the reagents were diluted in phosphate-buffered saline at pH 7.4, O.IM, 0.9% NaCl, 0.1% gelatin. The tubes were incubated overnight at 4 C. Separation of the free fraction from that bound to the antiserum was carried out at 0 C by the addition of 1 ml charcoal-dextran. After incubation for 12 minutes in a melting ice bath, the tubes were centrifuged for 15 min. at 2000 g. The supernatant (bound fraction) was transferred to scintillation vials and counted in a liquid... 

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