Staining and Detecting Electrophoresis Bands

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

There is often a need for a visualization procedure that is specific for a certain biomolecule, for example, an enzyme. If the enzyme remains in an active form while in the gel, any substrate that produces a colored product could be used to locate the enzyme on the gel. Although it is less desirable for detection, the electrophoresis support medium may be cut into small segments and each part extracted with buffer and analyzed for the presence of the desired component. 

Nucleic acids are visualized in agarose and polyacrylamide gels using the fluorescent dye ethidium bromide. The gel is soaked in a solution of the dye and washed to remove excess dye. Illumination of the rinsed slab with UV light reveals red-orange stains where nucleic acids are located. 

Ethidium bromide must be used with great care as it is a potent mutagen. Gloves should be worn at all times while using dye solutions or handling gels. 

Because of concerns about the safety of radioisotope use, researchers are developing fluorescent and chemiluminescent methods for detection of small amounts of biomolecules on gels. One attractive approach is to label biomolecules before analysis with the coenzyme biotin. Biotin forms a strong complex with enzyme-linked streptavidin. Some dynamic property of the enzyme is then measured to locate the biotin-labeled biomolecule on the gel. These new methods approach the sensitivity of methods involving radiolabeled molecules, and rapid advances are being made. 

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Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

By separating biochemicals on the basis of charge, size, and conformation, electrophoresis can provide valuable information, such as purity, identity, and molecular weight. Purity is indicated by the number of stained bands in the electropherogram. One band usually means that only one detectable component is present that is, the sample is homogeneous or electrophoretically... 

Figure 7.10 Agarose gel electrophoresis patterns for Ce(iv)/EDTA-induced site-selective hydrolysis of double-stranded DNA by Ce(iv)/EDTA and pcPNA additives. Bands were detected by staining with GelStar. (a) Site-selective hydrolysis of linearized PBR322 using pcPNAs. Lane 1, control lane 2, Ce(iv)/...

Bands of molecules separated by electrophoresis can be detected in the gel by various staining and destaining methods, such as the above mentioned ethidium bromide. However, more specific information can be obtained by application of blotting techniques. There are three different blotting techniques based on whether DNA, RNA, or protein is being analyzed. However, all blotting techniques have major steps in common. These include (1) the... 

Therefore, the movement of the ions within the gel is directly proportional to the applied voltage, and inversely proportional to the size of the protein. Gel electrophoresis is run for a fixed amount of time, allowing the small proteins to migrate further than the larger ones, resulting in their separation based on their size. Coomassie blue or silver staining can be used to detect the bands across the gel to confirm the presence of proteins with known molecular masses. 

In vitro translations were carried out according to the manufacturer (Promega Biotech) with [35S] methionine (1000 Ci/ml). After 1 hour incubation at 30 C, 1 jul-aliquots of the translation mixtures were used to determine hot trichloroacetic acid (TCA) precipitable incorporation of [35S] methionine. Products were analized by SDS-electrophoresis (SDS PAGE)(4). Gels were stained and dried, and labelled bands were detected by autoradiography. 

After electrophoresis, protein bands are transferred onto a solid support. Many aspects of a transfer can affect antigen detection. Some of these factors are specific to the transfer method and choice of membrane, whereas others apply to the entire blotting procedure. For example, the transfer efficiency may be affected by the presence of SDS in the gel and whether the gel was stained prior to transfer. 

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. 

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