Proteins and Nucleic Acids by Electrophoresis

CHAPTER 4 Characterization of Proteins and Nucleic Acids by Electrophoresis 111... 

Disc gel electrophoresis yields excellent resolution and is the method of choice for analysis of proteins and nucleic acid fragments. Protein or nucleic acid bands containing as little as 1 or 2 ju,g can be detected by staining the gels after electrophoresis. 

While chromatographic techniques have both analytical and preparative applications, electrophoresis is overwhelmingly an analytical tool. However, this does not mean that it is less important it is an indispensable technique across a broad range of the biosciences, particularly in analytical studies of proteins and nucleic acids (Hames 1998 Rickwood and Hames 1990). Electrophoresis has also been used to characterise other charged species, but these studies have been largely superseded by chromatography. Our present discussion of electrophoresis focuses on its core role in investigations of proteins and nudeic adds. 

This heat must be dissipated by cooling, which can be done but only to a limited extent The ability to dissipate heat efficiently is usually the factor that limits the speed of electrophoresis, since excess heat leads to non-uniform electrophoresis and a decrease in resolution. The main reason for this is convection in matrix-free electrophoresis in solution, and the effect of temperature on viscosity and diffusion. High temperatures can also lead to denaturation of proteins and nucleic acids. The thinner the layer used for electrophoresis, the more readily is the heat dissipated, and the higher the voltages that can be used. The thickness of the layer will be a compromise between a desire to have a thin layer to minimise heat problems whilst maintaining sufficient capacity to ran samples that can be detected easily. Consis-... 

For the determination of metal cations, chelates are created with oxine (8-hydroxyquinoline), alizarine or benzoin, then extracted by organic solvents. In biochemistry, fluorescence has numerous applications for the quantification of proteins or nucleic acids by means of reagents which can affix with specificity to these compounds. This approach, sometimes very elaborate, in association with electrophoresis constitutes a more sensitive and less restricting method than detection by radioactive substrates. 

SDS-Polyacrylamide Gel Electrophoresis Because many proteins or nucleic acids that differ In size and shape have nearly identical charge mass ratios, electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths. However, successful separation of proteins and nucleic acids can be accomplished by electrophoresis In various gels (semisolid suspensions in water) rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. When a mixture of proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel than do larger proteins. 

Capillary gel electrophoresis (CGE) aids in the separation of macromolecules such as proteins and nucleic acids whose mass-to-charge ratios do not vary much with size. In CGE, the capillary is filled with sieving material in the form of a gel or viscous polymer larger molecules are held up by the gel while small molecules pass through easily (this is the opposite to the SEC mode in HPLC ). Under well-controlled conditions, the mobility of the analyte is inversely proportional to analyte size. 

Nitrocellulose membranes must, prior to electrophoresis, be treated for 5 min with 2% Tween 60 in a veronal/citrate/oxalate buffer. Nitrocellulose membranes exhibit a selective absorptivity for proteins and nucleic acids which can be deliberately made use of. Detergent-treated nitrocellulose membranes do not absorb proteins at all and can be used at high potential gradients (up to 20 V/cm, for rather long periods of time (10-15 min). The best detection is effected by nigrosine staining. 

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