Equilibrium gel filtration

Figure 6.4 Experimental curves for equilibrium gel filtration, (a) The 1-mL tuberculin syringe was incubated in 109-pAf[14C]valine (O) or 109-/zM[14C]valine and 4-mM ATP ( ). Then 100 fjL of a solution of 26-fiM valyl-tRNA synthetase was added to the same solution. Stoichiometries of 0.8 and 1.1, respectively, were found for the binding of the amino acid. Note the return to baseline between the peak and the trough— the mark of a good equilibrium gel filtration experiment, (b) An artifact-induced double peak obtained from the binding of [y-32P]ATP and valine to the enzyme. Some of the labeled ATP hydrolyzed to [32P]orthophosphate, which traveled down the column faster than the fy-32P]ATP did.

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