Other Protein Quantitation Methods

Most potentiometric electrodes are selective for only the free, uncomplexed analyte and do not respond to complexed forms of the analyte. Solution conditions, therefore, must be carefully controlled if the purpose of the analysis is to determine the analyte s total concentration. On the other hand, this selectivity provides a significant advantage over other quantitative methods of analysis when it is necessary to determine the concentration of free ions. For example, calcium is present in urine both as free Ca + ions and as protein-bound Ca + ions. If a urine sample is analyzed by atomic absorption spectroscopy, the signal is proportional to the total concentration of Ca +, since both free and bound calcium are atomized. Analysis with a Ca + ISE, however, gives a signal that is a function of only free Ca + ions since the protein-bound ions cannot interact with the electrode s membrane. 

The feasibility of this approach to not only differentiate pathogenic and nonpathogenic strains of bacteria based on significant differences in protein mass but also on the basis of variations in levels of protein expression was demonstrated using a method for quantitating protein expression by LC/MS of whole proteins.54 This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these co-extracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to a marker protein, or internal standard, a relative ratio is obtained. This ratio... 

Like by other quantitative methods pure proteins give different results when the same weight was used, e.g., ovalbiunin 93%, rabbit IgG 90%, mouse IgG 80%, human IgG 97%, and chymotrypsinogen 100% when compared with BSA (data from Pierce Protein Assay Technical Handbook 1996). 

Upon use of structurally modified variants as internal standards for the particular analytes, the relative quantificahon of oligonucleotides, peptides, and small proteins was demonstrated [44]. The potential of the ILM to allow quantitative analyses of peptides without the use of internal standards was presented recently [43]. Linear correlahons between peptide amount and signal intensities could be found upon applicahon of increased matrix-to-analyte ratios between 25,000 and 250,000 (mokmol). The dynamic range of linearity thus spanned one order of magnitude. Unfortunately, the importance of the M/A ratio prevents the use of this method in samples with unknown orders of concentration, for example, in a proteomics environment. On the other hand, the method is applicable for the screening of enzyme-catalyzed reactions because the starting concentrahons of the peptides are generally known in such assays. 

All biochemical laboratory activities, whether in education, research, or industry, are replete with techniques that must be carried out almost on a daily basis. This chapter outlines the theoretical and practical aspects of some of these general and routine procedures, including use of buffers, pH and other electrodes, dialysis, membrane filtration, lyophilization, centrifugal concentration, and quantitative methods for protein and nucleic acid measurement. 

In general, it is very difficult reliably to extract and quantitate multiple vitamins from complex food systems, due to their diverse physical and chemical properties. Consequently, the extraction of the vitamins from the food matrix is usually the greatest challenge of vitamin analysis. This is especially true for the naturally occurring vitamins, which are often bound to other food constituents, such as carbohydrates or proteins. To prevent vitamin degradation or loss, the extraction conditions should complement the labile nature of the vitamins. Indiscriminate mixing and matching of extraction and quantitation methods is not recommended, since the extraction conditions can affect subsequent separation and quantitation steps. 

Electronic complexity reduction may provide an alternative method for sequence enrichment that is rapid, user-friendly and potentially quantitative. The device used in this experiment permits very high current densities and thus allows transport in buffers other than those typically used for electrophoresis. Beyond the use in complexity reduction, this device, with its ability to sustain high current densities, may have application in hybridization assays with a limited number of probes, immunoassays or other protein-binding reactions, and cell transport studies. Furthermore, the use of electrophoretic transport through all of the steps from sample processing through the assay should facilitate systems integration. 

Albumin can be measured quantitatively by the bromcresol green method in most species (Evans and Duncan 2003). Other proteins (described below) are measured by immunometric methods. Newer methods based on proteomics technology (concentration of proteins by acetone precipitation or ultracentrifugation, separation by 2-d gel electrophoresis or chromatographic techniques with subsequent identification and quantitation by mass spectrometry) have been used experimentally (Bandara and Kennedy 2002 Chapman 2002 Thongboonkerd et al. 2002a, b). 

Quantitative precipitin tests were performed as described.73 The amounts of precipitates obtained at the various concentrations were measured by the protein phenol method.74 The inhibition values are recorded in Table I. These inhibition data show that the precipitin reaction between the tetrahet-eropolysaccharide and the anti-GlcA antibodies is strongly inhibited by d-glucuronic acid, but with the exception of galacturonic acid none of the other carbohydrates were inhibitory. The data in the table also show that the carbohydrates tested did not inhibit the precipitin reaction between the polysaccharide and anti-GlcA-Rha antibodies. The monosaccharides alone cannot completely fit the active site of this antibody to give a precipitin test. 

For many years, the facility of the hydrochloric acid assay in gastric juice and the unavailability of good quantitative techniques for fractionation of proteins and mucosubstances have directed the interest of researchers toward the study of hydrochloric acid in the stomach. The only other material in gastric juice, studied and quantitated for many years and representative of the nondialyzable gastric secretory products, was pepsin. Study of other large molecular components has been hampered by the complexity of gastric juice, technical diflSculties encountered in its fractionation, and lack of adequate quantitative methods for the assay of these materials. 

CDT is also assayed extensively, especially in Europe, for detection of alcohol abuse. Other proteins, such as tti-acid glycoprotein, are carbohydrate deficient in this case as well. The gold standard for assessment is HPLC, although both capillary electrophoresis and immunoassays are more commonly used in clinical settings. Immunoassays in particular are poorly standardized, both qualitatively and quantitatively positive tests should be confirmed by an alternative method." Genetic variants of Tf may also complicate interpretation of results. 

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