5蛋白质检测技术.pptVIP

检测 Estimation of Protein Concentrations Biochemists are often interested in knowing the protein concentration in various preparations of biological origin. Cell extracts are complex mixtures that typically contain protein molecules of many different molecular weights, so the results of protein estimations cannot be expressed on a molar basis. It should be expressed as mass,such as g,mg,μg,ng. Most of their chemical properties vary with their amino acid composition. The properties of proteins are mainly based on their amino acid composition. for example, nitrogen氮 or sulfur硫 content or the presence of aromatic芳香族, hydroxyl羟基, or other functional groups. （1）Ultraviolet Spectra紫外光谱 Twenty different amino acids are commonly found in proteins. All 20 of the common amino acids are а-amino acids. They differ from each other only in their side chains侧链, or R groups, which vary in structure, size, and electric charge. None of the amino acids absorbs light in the visible region of the electromagnetic spectrum. However,all absorb in the infrared region. Only the aromatic amino acids芳香族氨基酸 phenylalanine, tyrosine, and tryptophan exhibit significant ultraviolet absorption above 250 nm. (Phe:275nm,Tyr:257nm, Trp:280nm). These strong absorptions can be used for spectroscopic determinations of protein concentration . Because most of proteins contain these three amino acids. This accounts for the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm. This is a specific property exploited by researchers and is often used in the characterization of proteins. （2）Assays Based on Dye Binding 1） Coomassie Brilliant Blue G-250考马斯亮蓝 Several other protocols for protein estimation enjoy prevalent usage in biochemical laboratories. The Bradford assay is a rapid and reliable technique that uses a dye called Coomassie Brilliant Blue G-250, which undergoes a change in its color upon non-covalent binding非共价结合to proteins. The binding is quantitative 定量an