Slab gel system

The other version of polyacrylamide gel electrophoresis is the slab gel system (Fig. 

The photopolymerization of the spacer gel is effected, the comb is carefully removed (in order not to destroy the surface of the wells), the plates are mounted into the electrophoretic apparatus, and samples are placed in the wells. After having filled the electrode vessels with buffer the electrophoretic separation can be started. Diffusion staining and destaining of the gel is done after disassembling the plates. 

On the contrary to rod shaped gels, slab gels can be dried on a paper sheet after staining and destaining and stored in this rather conventional form. Commercial instruments for slab gel electrophoresis and the destaining accessories can be obtained from e.g., Pharmacia Fine Chemicals, Uppsala, Sweden Desaga, Heidelberg, FRG or Bio-Rad Laboratories, Richmond, CA, USA. 

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Therein arose a new concern. How were laboratories going to be able to handle the bulk of samples received in a timely manner An analytical technique was needed that could provide as good or better results than those obtained via slab gel systems in a standardized and automated fashion. This technique was to be CE. 

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) use a Mini-Protein II slab-gel system (Bio-Rad) or equivalent. 

Righetti and co-workers [11] were one of the first to demonstrate the utility of classical isoelectric focusing for the chiral separation of small molecules in a slab gel configuration. In their system, dansylated amino acids were resolved enan-tiomerically through complexation with (i-cyclodextrin. Preferential complexation between the cyclodextrin and the derivatized amino acid induced as much as a 0.1 pH unit difference in the pK s of the dansyl group. 

There has been interest in miniaturizing and automating electrophoresis of proteins. Ruchel (1997) reported a miniaturized system where proteins are first separated by isoelectric focusing in millimeter diameter tubes. The tube s contents are transferred to a slab gel that is a few centimeters on a side. This technology was used to separate the proteins from a single giant neuron from Aplasia califomicus. More recently, native fluorescence has been used to resolve 200 proteins from a similar miniaturized electrophoresis system (Sluszny and Yeung, 2004). 

Figure 10.1 An illustration of a vertical slab-gel apparatus. Samples are applied in the wells set in the gel using a comb. Side-by-side comparison of samples is possible in slab systems (ref. 7). (Reprinted with permission from reference 7, Figure 11.2, Page 295, copyright (1994) Springer-Verlag.)...

The separation can be done in vertical or horizontal apparatus in rods or slabs. Because of the ease of use, horizontal slab electrophoresis systems are most commonly used in which the whole gel is submerged in buffer (Figure 13.8). 

A schematic representation of a CE system is presented in Figure 9.1. In this diagram, the CE components have obvious counterparts to those found in slab gel electrophoresis. Instead of buffer tanks there are two small buffer reservoirs, and the capillary takes the place of the gel (or more accurately, a gel lane). The capillary is immersed in the electrolyte-filled reservoirs, which also make contact with the electrodes connected to a high-voltage power supply. A new feature to the conventional gel electrophoresis format is the presence of an online detection system. 

Vertical slab-gel electrophoresis system that allows exact and constant temperation of the gel through circulation of temperated buffer around both sides of the gel. Specialised systems are available from several companies. 

PAGE analysis of protein from different samples was carried out in slab gels using 6% (w/v) polyacrylamide with the buffer system of Laemmli (12). After electrophoresis, proteins were visualized in the gels by Coomassie Blue R-250 staining (Sigma). 

Capillary gel electrophoresis (CGE) with polymer solutions is about 8 to 10 times faster than slab gel electrophoresis. However, the single-lane nature of CE was unable to compete in throughput with slab gel instruments, which are run in parallel. This led to the development of capillary array electrophoresis (CAE) [38] in 1992. As the name suggests, electrophoresis is performed in an array of capillaries to run multiple samples in parallel. Figure 8.20 shows a microfabricated capillary array system [39] on a glass wafer consisting of 96 channels. 

The electrophoretic separation technique is based on the principle that, under the influence of an applied potential field, different species in solution will migrate at different velocities from one another. When an external electric field is applied to a solution of charged species, each ion moves toward the electrode of opposite charge. The velocities of the migrating species depend not only on the electric field, but also on the shapes of the species and their environmment. Historically, electrophoresis has been performed on a support medium such as a semisolid slab gel or in nongel support media such as paper or cellulose acetate. The support media provide the physical support and mechanical stability for the fluidic buffer system. Capillary electrophoresis (CE) has emerged as an alternative form of electrophoresis, where the capillary wall provides the mechanical stability for the carrier electrolyte. Capillary electrophoresis is the collective term which incorporates all of the electrophoretic modes that are performed within a capillary. 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

The combination of horizontal slab-gel electrophoresis and in situ assay of enzyme activity is a versatile and powerful method for detecting protein variants (isozymes and allozymes) in plants. Such variants are potentially useful as genetic markers for mapping chromosomes and in studies of breeding systems,1 population structure,2,3 gene flow,4 polyploidy,5,6 and systematics.7-9... 

A high molecular weight, citrate-inducible, outer membrane polypeptide has been detected by slab gel electrophoresis (65). This is believed to be the receptor for the ferric citrate transport system earlier characterized by Frost and Rosenberg (60). 

CIEF is the replication in the capillary of slab gel isoelectric focusing, a widely used separation mode, particularly suitable for protein separation. In isoelectric focusing, substances are separated by applying an electric field in a buffer system forming a pH gradient Analytes focus where the buffer local pH equals their isoelectric points. 

Selection of the appropriate counterion is also important. Lithium ion has the lowest mobility of the alkali earth metals. Its use provides for a low-conductivity buffer. Sodium salts are used more frequently due to purity and availability. It makes little sense to ever use a potassium salt. Dual-buffering systems with low-mobility ions and counterions (Tris-phosphate, Tris-borate, aminomethylpropanediol-cacodylic acid) are effective in minimizing buffer conductivity. These buffers are often used in the slab-gel, where low conductivity is particularly important. 

Because CE uses online optical detection, artifacts can result in the form of system peaks. These often originate from the sample or the interfaces between the sample and the separation buffer because any species that absorbs at the detection wavelength wfll generate a response. This differs from protein slab gel electrophoresis where detection specificity is governed by a protein specific stain. It is not uncommon, for example, for buffer species present in the sample but not in the separation buffer to generate system peaks. However, clinical serum protein electrophoresis provides one example where artifacts are elimmated by CE. 

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