Species protein analysis

A calibration curve shows the response of an analytical method to known quantities of analyte.8 Table 4-7 gives real data from a protein analysis that produces a colored product. A spectrophotometer measures the absorbance of light, which is proportional to the quantity of protein analyzed. Solutions containing known concentrations of analyte are called standard solutions. Solutions containing all the reagents and solvents used in the analysis, but no deliberately added analyte, are called blank solutions. Blanks measure the response of the analytical procedure to impurities or interfering species in the reagents. 

Quantitative protein analysis is accomplished by combining protein separation, most commonly by high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE), with MS-(mass spectrometry) based or tandem MS (MSZMS)-based sequence identification of selected, separated protein species. This method is sequential, labor intensive and difficult to automate. It selects against specific classes of proteins, such as membrane proteins, very lar ge and small proteins, and extremely acidic or basic proteins. Tlie technique has a bias toward highly abundant proteins, as lower abundance regulatory proteins (e.g., transcription factors and protein kinases) are rarely detected when total-cell lysates are analyzed. 

Although the analysis of the infrared spectra involves several species proteins, galactolipids, Chi, carotenoids, etc., we show in this ir study that beside the prominent protein bands some Chi bands can be identified. 

Usually isoelectric focusing on a thin gel is chosen for die first dimension separation. Once the ionic species (protein) are focused into narrow bands, they serve as zones for the second-dimension analysis. The second dimension can be any of the following immunoelectrophoresis, a discontinuous SDS-polyacrylamide gel system, or gradient electrophoresis. 

The main challenge when applying this method for protein analysis is the lack of commercial isotopically labeled proteins. Thus, most applications are focused on small molecules, such as organic mercury, organic tin and so on. However, there is increasing interest in the use of the ICP-MS linked system and species-specific isotope dilution for quantification of peptides or proteins due to the outstanding performance of ICP-MS. 

Although fluorimetric analysis of biological systems is experimentally simpler and less disruptive to inherent chemical properties when the use of synthetic fluorescent molecules can be avoided, in many instances the intrinsic properties of biological chemicals do not provide sufficient means for characterizing or quantifying desired properties. In the case of protein analysis, for example, many species lack Trp or Tyr residues altogether in protein molecules that do contain these amino acids, the fluorescence characteristics may be inadequate to yield the needed information. 

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