Analysis of Binding Data

A last resort is sometimes switching the species used as the source of the binding protein. The amount of binding protein per milligram of total protein (and thus the signal-to-noise ratio) is sometimes markedly different between even closely related species. 

This chapter may contain too many formulas for the average taste. The formulas are supposed 

The mathematical treatment of binding data falls into two topics. 

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Most laboratories now have access to powerful computers and an extensive array of commercially available data analysis software (e.g., Prism (GraphPad, San Diego, CA), Sigma Plot (San Rafael, CA)). This provides ready access to the use of nonlinear regression techniques for the direct analysis of binding data, together with appropriate statistical analyses. However, there remains a valuable place for the manual methods, which involve linearisation, particularly in the undergraduate arena and these have been rehearsed in the above text. 

A number of useful reviews of small molecule-protein interaction have appeared (D2, E4, S26, W7), and these contain detailed treatments of the mathematical analysis of binding data as well as information on experimental methods. 

Both equations are used in the linear and graphical analysis of binding data. 

Analysis of binding data depends on the selection of a model. Since the binding is likely to be nonspecific, sharing also the feature of a ligand binding to a lattice, the models developed for adsorption onto surfaces or lines... 

Fig. 7. I-SDF-1 a and IIIB gp 120 heterologous competition on hNT neurons. hNT cells were incubated with radiolabeled SDF-la or niB gpl20 in the presence and absence of varying concentrations of unlabeled protein. (A) I-SDF-1 a is displaced by unlabeled IITR gpl20. Scatchard analysis of binding data (inset), = 70 nM. (B) gpl20 is dis-...

Analysis of binding data Computer program Sigmaplot 5 0 (Jandel, Corte Madera, CA)... 

Figure 4 shows an analysis of binding and dissociation of FLPEP in permeabilized cells in the absence (top panel) and the presence of GTPyS (lower panel). Dissociation is initiated, as indicated, by the addition of a receptor antagonist, tBoc-phe-leu-phe-leu-phe. The solid line is a fit to the data. The data from this experiment have... 

Preliminary inspection and analysis of the data to try to establish a model that adequately describes the binding. For example, multiple components or cooperativity may be identified. 

To make contact with atomic theories of the binding of interstitial hydrogen in silicon, and to extrapolate the solubility to lower temperatures, some thermodynamic analysis of these data is needed a convenient procedure is that of Johnson, etal. (1986). As we have seen in Section II. l,Eqs. (2) et seq., the equilibrium concentration of any interstitial species is determined by the concentration of possible sites for this species, the vibrational partition function for each occupied site, and the difference between the chemical potential p, of the hydrogen and the ground state energy E0 on this type of site. In equilibrium with external H2 gas, /x is accurately known from thermochemical tables for the latter. A convenient source is the... 

Kinetic studies were attempted at 4, 15, 25 and 37 °C, but the colloids tended to aggregate at all temperatures above 4°C. Four models were used to determine the binding mechanisms from the kinetic data. A detailed analysis of binding at 4 °C, was made. Models were set up involving one or two surface sites which also satisfied the overall kinetics but the analyses were not definitive. Although it was demonstrated that the cells were capable of endocytosis of fluorescence-conjugated transferrin, there was no evidence for the endocytosis of the cationic colloids. 

Binding of heme by isolated N-domain causes a change in sedimentation coefficient consistent with a more compact conformation and leads to the more avid association with the C-domain (125). Sedimentation equilibrium analysis showed that the Kd decreases from 55 pM to 0.8 pM (Fig. 5) (106). In addition, the calorimetric AH (-1-11 kcal/mol) and AS (-1-65 kcal/mol K) for the heme-N-domain-C-domain interaction and the AH (-3.6 kcal/mol) and AS (-1-8.1 kcal/mol K) derived from van t Hoff analysis of ultracentrifuge data for the interaction in the absence of heme indicate that hydrophobic interactions predominate in the presence of heme and a mix (e.g., hydrophobic and van der Waals forces) drives the interaction in the absence of heme. However, FTIR spectra (Fig. 6) indicate that little change in the secondary structure of domains or intact hemopexin occurs upon heme binding (104). 

With these caveats in mind, a number of points support a relatively short distance for guanine-Ru(bpy)3 electron transfer in our system. First, as shown in Fig. 3, when the guanines are scarce in the sequence, the binding of Ru(bpy)3 at sites on the helix to which electron transfer is not efficient must be considered [19]. Thus, the oxidant can bind to the duplex at sites too far from a guanine for electron transfer. Quantitative analysis of these data suggests that electron transfer occurs within distances that are consistent with those observed in studies of covalently bound donors and acceptors and therefore with values of the tunneling parameter fi consistent with those observed in other systems [38, 50, 98-100]. 

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