Diagonal electrophoresis

An effective way to isolate these fragments is through diagonal electrophoresis (Figure 5.26) (the basic technique of electrophoresis is described in... 

Perham, R.N., and Jones, G.M.T. (1967) The determination of the order of lysine-containing tryptic peptides of proteins by diagonal paper electrophoresis. Eur. J. Biochem. 2, 84-89. 

Holloway, J. W. Beghe, B. Turner, S. Hinks, L. J. Day, I. N. Howell, W. M. Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE). Hum. Mutat. 1999,14(4), 340-347. 

Diagonal electrophoresis and its relative diagonal chromatography are useful for other purposes as well. After electrophoresis or chromatography is conducted in one direction, the paper or thin-layer plate may be sprayed with a reagent that will react with some components or may be irradiated with light before the separation is repeated in the second direction (Fig. 3-5).102/26°... 

This mixture is subjected to electrophoresis in the perpendicular direction under the same conditions as those of the first electrophoresis. Peptides that were devoid of disulfides will have the same mobility as before, and consequently all will be located on a single diagonal line. In contrast, the newly formed peptides containing cysteic acid will usually migrate differently from their parent disulfide-linked peptides and hence will lie off the diagonal. These peptides can then be isolated and sequenced, and the location of the disulfide bond can be established. 

Figure 4.27. Diagonal Electrophoresis. Peptides joined together by disulfide bonds can be detected by diagonal electrophoresis. The mixture of peptides is subjected to electrophoresis in a single lane in one direction (horizontal) and then treated with performic acid, which cleaves and oxidizes the disulfide bonds. The sample is then subjected to electrophoresis in the perpendicular direction (vertical).

The positions of disulfide bonds can be determined by diagonal electrophoresis (p. 96). The disulfide pairing is unaltered by the mutation if the off-diagonal peptides formed from the native and mutant proteins are the same. 

Nestler, H.P. and Doseff, A. (1997) A two-dimensional, diagonal sodium dodecyl sulfate polyacrylamide gel electrophoresis technique to screen for protease substrates in protein mixtures. Anal. Biochem. 251, 122-125. 

The technique of two dimensional electrophoresis with two identical procedures applied in a right angle to each other, does not offer additional resolution and would be therefore useless unless there is some reaction that alters the charge of some members in the mixture of separated components performed between individual runs. Then the reacted members would deviate from the diagonal where all the other non-reacted components would be localized. An example of this procedure is that of MikeS and HolySovsky [19] and Hartley et al. [20]. Using this procedure disulphide peptides from a tryptic hydrolysate of an enzyme were analyzed. The hydrolysate is separated in the first run on a filter paper strip. The separated peptides are then oxidized with performic acid vapour by means of which the S-S bridges are broken... 

Crossed electrophoresis with two currents acting perpendicularly was described by McDonald and Urbin [24]. The electrophoretic chamber has four electrodes, two of which are positive, two are negative. After the potential has been applied, the substances to be separated start to move along the diagonal. Therefore this technique is sometimes referred to as continuous diagonal electrophoresis. The technique was applied to the separation of DNP amino acids. 

Edman degradation (p. 92) phenyl isothiocyanate (p. 92) cyanogen bromide (CNBr) (p. 94) overlap peptides (p. 95) diagonal electrophoresis (p. 96)... 

Milner PA, Gogel G, Barber J. Investigation of the spatial relationships between Photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis. Photosynth Res 1987 13 185-198. 

Compare the diagonal electrophoresis (textbook, p. 96) patterns obtained with the normal and the mutant proteins. If these patterns are essentially identical, the disulhde pairing is the same in both proteins if they are not, the new cysteine residue is probably involved in a new disulfide pairing in this mutant. 

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