High voltage paper electrophoresis

High voltage electrophoresis (2000-4000 V) on Whatman 3 MM paper (57 cm in length) is carried out in an apparatus in which the ends of the paper are immersed in the two buffer chambers and the rest of the paper is supported on a rack that is completely immersed in an inert solvent (e.g. Varsol), cooled by circulating tap water. In our laboratory we use a Savant apparatus, but others are also suitable. 

The origin is generally marked on the paper (with pencil) at 3j inches from one end for electrophoresis at pH 1.9 and at 9-10 inches for electrophoresis at pH 3.6, 4.7 or 6.5, although certain procedures may require a shift in the position of the origin. Samples are usually applied to the dry paper, and if volumes larger than 10-20 pi are to be applied at the same point, the paper should be dried (with a hair dryer or other type of fan) between applications. In some instances, the samples (in less than 25 pi) may be applied directly to papers saturated with the electrophoresis buffer (after air drying for 5-10 min). If the samples are applied to dry papers, the papers are then placed on the electrophoresis racks and saturated with a stream of buffer (a squeeze bottle is convenient) in such a way that the solvent will approach the sample areas by capillary action at about the same rate from both sides. 

After electrophoresis for the appropriate time, the papers are placed on glass rods in an oven at 60°C (the oven should preferably be equipped with a fan venting to a fume hood or to the outside) and dried (usually requires 20-30 min). They are then dipped in a ninhydrin reagent (see p. 183) and again heated in the oven for 15-60 min for color development. 

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Enzyme and substrate Cleavage shown by high voltage paper, electrophoresis [Ref. (3)] V max K (M)... 

Phenyl isothiocyanate has also been utilized to analyze small quantities of peptides or short-chain peptides (88) by carrying out sequential degradation with detection by dansylation. Using dansyl-Edman degradation, Leadbeater and Bruce-Ward (60) identified 60 tryptic peptides of/8-casein that had previously been separated by high-voltage paper electrophoresis. 

K4. Kickhofen, B., High voltage paper electrophoresis. In Paper Electrophoresis, Ciba Foundation Symposium (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 206-209. Churchill, London, 1956. 

The enzymatic radioassay method for the analysis of acetylcholine and choline in brain tissue has been reported by Reid et al. [210]. The method describes the determination of nanogram amounts of acetylcholine and choline in as little as 10 mg of brain tissue, involves isolation of acetylcholine by high-voltage paper electrophoresis, alkaline hydrolysis of acetylcholine to choline, and conversion of this into [32P]-phosphoryl choline in the presence of choline kinase and [y32P] ATP. The labeled derivative is isolated by column chromatography on Bio-Rad AG1-X8 resin, using Tris buffer solution as the eluent. Cerenkov radiation from 32P is counted (at 33% efficiency) in a liquid scintillation spectrometer. The amount of phosphorylcholine is proportional to the amount of choline over the range of 0.08-8.25 nmol. 

After hydrolysis is complete the tubes are cooled, scored and opened, and the acid is removed, either by rotary evaporation or in a heated desiccator over NaOH at 40-50°C. In our laboratory it is standard procedure to examine 1/10 of each hydrolysate by high-voltage paper electrophoresis at pH 1.9 (see Appendix I) prior to analysis to ensure that appropriate amounts are analyzed. Some amino acid derivatives (oxidation products of methionine, methylated lysines, etc.) are also sometimes observed by this procedure. It is usually helpful to add an internal standard, either prior to hydrolysis to reveal hydrolytic losses or prior to analysis to reveal analytical losses. For this purpose we have used norleucine (0.03 pmoles). This amino acid elutes after leucine on the 60 cm column. 

Many derivatives of histidine are not stable to acid hydrolysis and are not discussed here (however, see 2.12.2 for those that occur naturally in proteins). Brief mention should be made of the iodination of histidyl residues by HOI ( 3.7.2). The mono- and diiodohistidines can be identified and distinguished from the iodinated tyrosines by high voltage paper electrophoresis in 1 M formic acid (Roholt and Pressman 1972) after complete enzymic hydrolysis of the protein or peptide ( 2.11 Roholt and Pressman 1972). Quantitation and identification are facilitated by the use of radiolabeled reagent. 

Figure 9.6. High-voltage paper electrophoresis with cooling.

A9. Atfield, G. N., and Morris, C. J., Analytical separations by high-voltage paper electrophoresis. Amino acids in protein hydrolysates. Biochem. J. 81, 606-614 (1961). 

ElO. Efron, M. L., High voltage paper electrophoresis. In Chromatographic and Electrophoretic Techniques. Vol. II. Zone Electrophoresis (I. Smith, ed.), 2nd Ed., pp. 166-193. Wiley (Interscience), New York, 1968. 

Jll. Juul, P., Quantitative measurement of individual free amino acids in urine by means of high voltage paper electrophoresis. Investigations of a group of mentally retarded patients. Scand. J. Clin. Lab. Invest. 18, 629-637 (1966). 

Abreviations Mv (malvidin-3-glucoside), HVPE (high voltage paper electrophoresis)... 

Ionisation constants of vitisin A and the various ethyl-linked malvidin-3-glucose / catechin / epicatechin dimers were determined by high voltage paper electrophoresis (HVPE) using chromatography paper (Chr. 1,. Whatman, England) as previously reported (28). All relative mobilities (Rm) were compared to anionic standards orange G and xylene cyanol. Fructose was used as... 

Amino Acids, Determination by High-Voltage Paper Electrophoresis... 

For these reasons we have used depurinating conditions (15 min, lOO C, 90 % HCOOH) which separated adenine, guanine and the platinum-DNA adducts from apurinic acid. These products were resolved by high voltage paper electrophoresis at pH 2. The paper was cut into 1 cm strips which were eluted in water and the profile of platinum concentration was determined by flameless atomic absorption. Under these conditions, the apurinic acid migrated toward the positive electrode while the purine bases and the adducts migrated toward the negative electrode. For all three compounds, less than 10 % of the platinum was detected on the apurinic acid, even at r 0.4. The electrophoresis profiles of hydrolysed platinum-DNA complexes formed by eis-DDP, ii ans-DDP and... 

The separation of organic acids and especially of the aliphatic ones by high voltage paper electrophoresis has been the aim of a number of workers during the last 10 years (Bll, B12, G18, G19, G20, M23, W15). 

G18. Gross, D., High-voltage paper electrophoresis of non volatile organic acids and their mixture with amino acids. Nature 178, 29 (1956). 

G19. Gross, D., Two-dimensional high-voltage paper electrophoresis of amino and other organic acids. Nature 184, 1298 (1959). 

Equipment for low and high voltage paper electrophoresis Apparatuses used for paper electrophoresis consist of two electrode jars and a system that holds the paper sheet between them and prevents, at least partly, the imbedded buffer from evaporation. The equipment used could be categorized into two groups with either horizontal or vertical arrangement of the paper strip (Figs. 

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