Plotting binding data

Examples of these are shown for the saturation data in Figure 4.2. At first glance, these transformations may seem like ideal methods to analyze saturation data. However, transformation of binding data is not generally recommended. This is because transformed plots can distort experimental uncertainty, produce compression of data,... 

To construct the Hill plot (Figure 5.10E), it was assumed that fimax was 0.654 fmol/mg dry wt., the Scatchard value. The slope of the plot is 1.138 with a standard deviation of 0.12, so it would not be unreasonable to suppose % was indeed 1 and so consistent with a simple bimolecular interaction. Figure 5.10B shows a nonlinear least-squares fit of Eq. (5.3) to the specific binding data (giving all points equal weight). The least-squares estimates are 0.676 fmol/mg dry wt. for fimax and... 

A historically important graphical technique to describe binding data (see O Figure 10-2) is the Scatchard plot (1949). This is simply a linearization of the function ... 

Scatchard plot of the binding data shown inO Figure 10-la, illustrating how the Kd and R can be obtained from such a linearization procedure... 

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 plot of the measurable quantity ln 8/(l - 8) as a function of ln[02] should give a straight line with slope n. Such a plot is called the Hill plot and is widely used in reporting and interpreting experimental binding data. 

Two fmrther equations have been derived from Eq. (2), and these are frequently employed to plot experimental data in order to obtain an estimate of the number of binding sites (n) and the apparent association constant (fc). The reciprocal plot (K9) involves the following rearrangement of (2). 

The main plots used in enzyme kinetics and receptor binding studies are the Scatchard plot, the Lineweaver-Burk plot, and the linearization for estimation of the Hill coefficient. This chapter gives a short survey of these transformations of enzyme kinetics or receptor binding data. 

Perhaps the most widely used method for extracting binding data is the Scatchard plot. This is obtained from... 

While Scatchard plots may be useful to display the binding data, they are not a useful way to analyze them. It is usually preferred to fit the raw binding data with a nonlinear regression. Selection of the dependent and independent variables... 

Binding data is more traditionally presented after the manner of Equation 5.12 in the form of a Rosenthal plot (Figure 5.21).25... 

As a complement to question 1, plot the binding data of thiomuscimol in Michaelis-Menten (response versus [L]) format. Try to create a Lineweaver-Burk plot (1/response versus 1/[L]) and perform a linear regression on the data. What is the problem you encounter while making this graph Do your best to graph the data. From the best-fit line of the Lineweaver-Burk plot, determine Kt) and Enva. How well does Ku in this graph match the Ku you determined in... 

Rosenthal plot A Rosenthal plot is generated from ligand binding data and is useful for determining maximal specific binding (Bmax). 

Fig. 6.1. (a) Colchicine binding in Hymenolepis diminuta. (b) Scatchard plot of data taken from (a). (After Watts, 1981.)... 

Fig. 1. Assay for progestin receptors in human breast cancer cytosol. A. Saturation analysis. Total ( — ) and aspecific (o—o) binding are measured Specific binding (solid line) is calculated by subtraction. B. Scatchard plot derived from the binding data by the method of Chamness and McGuire [8]. Numerical data protein concentration of cytosol. 1.8 mg/ml assay volume, 100 /d cytosol volume, 50 /ul. Calculated values Kd = 0.76 nmol/l progestin receptor content, 510 fmol/mg protein.

Two other possible transformations of saturation binding data mentioned earlier are the Hofstee (B versus B/F) and Woolf Plots (F/B versus / ). As with the Scatchard equation, these equations can be derived from algebraic manipulations of the equations listed above. 

The AT-specificity in the binding of tilorone to DNA was also observed in the quantitative equilibrium binding measurements. The equilibrium binding data were plotted as r/(u) vs. r, where r is the moles of bound tilorone divided by the DNA concentration in base pairs60, and (u) is the concentration of unbound tilorone. 

Figures 13 to 17 show some experimental plots of titration data according to Eq. (14). For some of the examples chosen, ion binding data were available, so that the left-hand side of Eq. (14) could be plotted against Z.

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