Protein separation techniques extract preparation

Biochemists extract the contents of cells and separate the constituents based on differences In their chemical or physical properties, a process called fractionation. Of particular interest are proteins, the workhorses of many cellular processes. A typical fractionation scheme Involves use of various separation techniques In a sequential fashion. These separation techniques commonly are based on differences in the size of molecules or the electrical charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme Is designed so that each step yields a preparation with fewer and fewer contaminating proteins, until finally only the protein of Interest remains (Figure 1-22). 

Due to the fact that MT is a native protein, its separation from real samples is closely connected with an isolation process. This procedure is being imderstood as a complex of individual steps, which start with pretreatment methods involving extraction and purification and continue with separation techniques prior the MT detection. At the beginning of the quantification of MT, it is necessary to prepare samples (from cells or tissues) according the following protocol (Figure 3). In the case of the cells that were treated with metal ions, samples should be washed with buffer solution to remove residual culture medium and metals adsorbed on the surface of the cells. 

Over thirty different elements have been determined in medical and biological materials by atomic absorption spectroscopy. The popularity of the technique is due to a number of factors, including sensitivity, selectivity, and ease of sample preparation. With biological fluids, often no preparation at all is required. The techniques employed usually involve simple dilution of the sample with water or with an appropriate reagent to eliminate interference. Alternatively, the element to be determined is separated by solvent extraction. Either an untreated sample, a protein free filtrate, or an ashed sample is extracted. 

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by... 

One of the most crucial considerations in proteomic analysis is sample preparation because this will ultimately dictate the number and type of proteins that can be processed. The first priority is to establish the precise protein system to be studied [e.g., will this be a comprehensive and exhaustive catalogue of every expressed protein within a tissue or cellular extract, or is only a small subset of a cellular proteome (e.g., only phosphoproteins or membrane-bound proteins) sufficient for analysis ]. Whether a full or partial proteome, or even a limited number of specific proteins is required for analysis, it is crucial that the extraction technique provide maximal protein recovery while preserving the integrity of the protein complex to be examined. Furthermore, the method of preparation must be totally compatible with the separation methods to be used. This is particularly important for separation technologies that are reliant on protein-protein interactions or drug/ligand/antibody, etc. interactions. Poor recovery of proteins is clearly... 

Liquid-liquid partition chromatography techniques based on aqueous-aqueous systems have successfully been employed in the fractionation of crude human serum, purification of steroid hormone-binding proteins from human serum, isolation of basic proteins from crude bacterial extracts, purification of immunoglobulins and monoclonal antibodies, DNA fractionations by size, topology and base sequence, as well as the isolation of soluble and ribosomal RNAs in preparative amounts from bulky mixtures [10]. Highspeed CCC using PEG-dextran system has also been employed in the separation of proteins [6]. 

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