Binding data, analysis

Few selected software packages available for binding data analysis... 

Scatchard analysis, a common linear transformation of saturation binding data used prevalently before the widespread availability of nonlinear fitting software. The Scatchard transformation (see Chapter 4.2.1), while easy to perform, can be misleading and lead to errors. 

Figure 5. Guanine-nucleotide-dependent receptor interconversion from LRG" to LR. Penneabilized cells (10 /mL) (no GTPyS 1 ml Mg " ) were allowed to bind FLPEP for 1 min. Data analysis begins with the addition of antibody to fluorescein (after 60 s, but not indicated). At 90 s (indicated), GTPyS is added to induce interconversion to the rapidly dissociating state. The guanine nucleotide concentrations are shown (saturated at 10" If). The solid lines are the fit to the onestep model shown in the inset, and the k for the fit is shown at the end of each solid line. Data...

Fig. 37.7. PLS biplot derived from the biochemical binding data in Table 37.10, after log doublecentering and analysis by two-block PLS [56]. The reading rules are the same as those indicated with Fig. 37.6. Reproduced with permission of E.J. Karjalainen.

These problems are provided to afford an opportunity for the reader to analyze binding data of different sorts. The problems do not require nonlinear least squares analysis, but this would be recommended to those with access to appropriate facilities. It must be emphasized that, while linearizing transformations allow binding data to be clearly visualized, parameter estimation should... 

Munson, P. J. (1983). Experimental artifacts and the analysis of ligand binding data results of a computer simulation, J. Receptor Res., 3, 249-259. 

De Lean, A., Hancock, A. A. and Lefkowitz, R. J. (1982). Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes, Mol. Pharmacol., 21, 5-16. 

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. 

It is clear that this approach does not deliver information about the binding process at atomic resolution. Rather, it focuses on the binding affinities of ligands to the virus. Careful data analysis allows discrimination of residues that actively participate in the binding from those that contribute less to the interaction. Ligands in the intermediate exchange regime on the chemical... 

The data presented in Figs. 3 and 4 are examples of the types of kinetic binding data that are readily acquired with commercially available flow cytometers. Quantitative, real-time analysis of fluorescent A-formyl peptide association with neutrophil receptors has been described by Fay et al. (4), and this publication should be consulted for detailed protocols required for quantitative kinetic assays (see Notes 5 and 6). 

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