Calibration, protein expression data

A DICE experiment produces large amounts of data, or more exactly, volume values for thousands of gel spots. In order to make correct inferences from these data, statistical methods are quite important. Many of the statistical methods used in DNA microarray studies can be adapted for analyses of gel data. In this section we focus on the calibration and normalization of protein expression data as well as on the detection of differentially expressed proteins resulting from a DICE experiment. 

To detect ricin, we studied its inhibitory effects on luciferase expression by adding a series of concentrations of ricin into the IVT reactions in the array device. To achieve a lower detection limit, 4-hr protein expression was used, though ricin detection can be achieved in as short as 5 minutes. Ricin sample size was 2 pL. As shown in Figure 5a, the expression yield of luciferase, indicated by luminescence, decreases with the concentration of ricin (solid circles). However, the expression yield remained the same when the ricin is heat denatured and its toxicity is deactivated (open circles). The error bar of each data point indicates the standard deviation that is obtained from three repeat experiments. The calibration curve is obtained by plotting the detection... 

Gel permeation chromatography of protein linear random coils in guanidinium chloride allows simultaneous resolution and molecular weight analysis of polypeptide components. Column calibration results are expressed in terms of a log M vs. Kd plot or of effective hydrodynamic radius (Re/). For linear polypeptide random coils in 6M GuHCl, Re is proportional to M0 555, and M° 555 or Re may be used interchangeably. Similarly, calibration data may be interpreted in terms of N° 555 (N is the number of amino acid residues in the polypeptide chain), probably the most appropriate calibration term provided sequence data are available for standards. Re for randomly coiled peptide heteropolymers is insensitive to amino acid residue side-chain composition, permitting incorporation of chromophoric, radioactive, and fluorescent substituents to enhance detection sensitivity. 

The analytical data, expressed as frequency shift, are the differences in the frequency of the crystal before the addition of thrombin and after the washing with buffer subsequent to the affinity interaction. A signal generated by the aptamer-protein interaction is considered significative when the difference between the frequency values corresponding to the two buffers is higher than 3 Hz. Different concentrations of the protein can be used to build a calibration plot. An example of calibration plot is shown in Fig. 5. 

In choosing proteins for this calibration plot, we have avoided proteins known to deviate markedly from spherical shape. [An interesting extension of this approach would be the expression of data for non-spherical solutes using... 

Basically two ways of parametrization can be followed. Class I force fields like AMBER (Case et al. 2005) or GROMOS (2011), work with a simpler energy expression and their parameterization is based on experimental data. They find a wide application to proteins, nucleic acids, and carbohydrates, as well as their complexes. Class II force fields, e.g., the Merck Molecular Force Field (Halgren 1996) include higher order and cross terms, too they are calibrated to... 

---