Laboratory Analysis of Biomolecules

The newer applications involve the field of biotechnology. Proteins produced by genetically altered organisms such as bacteria must be examined to verify that they are identical to the same proteins produced by humans. Also, analysis of DNA from crime scenes is relatively recent. Indeed, DNA analysis and fingerprinting are powerful tools in modern forensics. 

As discussed in Chapter 11, electrophoresis refers to a group of techniques used to separate and study molecules with electrical charges. Based upon these charges, both sign and magnitude, biomolecules such as proteins, peptides, amino acids, nucleic acids, and fragmented nucleic acids migrate in an electrical 

A variety of buffers is used in electrophoresis. The selected buffer must contain ions to carry the current. Other than current-carrying capacity, the most critical criterion for buffer selection is the stability of the sample to be analyzed. Many proteins are unstable in acidic pHs, so alkaline buffers are frequently employed. Tris-(hydroxymethyl)amino methane (TRIS or THAM), sodium acetate, and ethylenedi-aminetetraacetate (EDTA) are common solutes in buffers, with pHs between 7.9 and 8.9 typical. (Refer to Chapter 5 for a discussion of buffers.) These buffers also work well with nucleic acid fragments. In addition, phosphate buffers, e.g., 10 mMK3P04, are often used with nucleic acid fragments (1.0 mM = 0.0010 M). 

This method is very useful for separating amino acids found in food samples. The most effective matrix for separation is an absorbent cellulose-based filter paper. A very effective mobile phase is 70% isopropyl alcohol in water. Although the 20 amino acids are chemically very similar, they may be successfully separated by this method. Amino acids interact with the stationary phase to different extents, thus moving at different speeds. Chemical differences among amino acids that determine migration speed include molecular weight, charge, and polarity. 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

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Bioanalysis may be defined as laboratory analysis of biomolecules. Biomolecules, in turn, are organic compounds with biological activity, generally important only in biological systems, or cells. Biochemistry is the study of structure and function of biomolecules. Biotechnology, a related concept, concerns the industrial applications of biochemical techniques. Thus bioanalysis, biochemistry, and biotechnology are closely related concepts, all concerned primarily with biomolecules. 

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