Paper electrophoresis apparatus

Figure 11.22 represents a paper electrophoresis apparatus. The soaked cellulose sheet is sandwiched between two horizontal glass plates with the ends dipped into vessels containing more electrolyte solution. The electrodes are also dipped into these vessels, as shown. The sample is spotted in the center of the sheet, and the oppositely charged ions then have room to migrate in opposite directions on the sheet. Qualitative analysis is performed much as with paper chromatography, by comparing the distances the... 

In a number of experiments studying the incorporation of S3504 into seromucoid fractions 10-/il. samples of plasma were applied to paper strips and subjected to electrophoresis in the Beckman-Spinco paper electrophoresis apparatus. The resultant oven-dried paper strips were attached to an 8 x 10 sheet of paper and applied directly to a sheet of Eastman No-screen x-ray film. Exposure extended over a period of 1 month. At the end of this time the film was developed in the usual manner (see Figure 2). The paper strips were then stained with bromophe-nol blue to demonstrate the major protein fractions. 

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. 

HCl aq EtOH). Recrystd from warm, acidified EtOH by addition of ammonia. The crude material (Ig) can be extracted with EtOH (50mL) in a Soxhlet apparatus for lOh to remove impurities. Impurities can be detected by paper electrophoresis. [Petrova et al. Anal Lett 5 695 1972.]... 

Put the plate on the cooling plate of a horizontal electrophoresis apparatus. Fill the buffer chambers with 1 1 diluted Soln. B (= electrode buffer) and connect the electrode buffer to the gel by filter paper bridges, wetted with electrode buffer. The filter paper covers the gel for about 5 mm. Remove carefully air bubbles between papers and gel. 

Apply some drops of kerosene on the cooling block of the horizontal electrophoresis apparatus and apply the IPG strips with the acidic side to the anode. Make 1 cm broad strips from 2 mm thick filter paper. Wet the strips with deionized water (no ultra-pure water ) and place them on top of the IPG strips as well as at the anodic (acidic) and the cathodic (basic) ends of the strip(s). 

The connection between electrode chambers of a horizontal electrophoresis apparatus filled with buffer A and the cellulose support is made by wetted paper bridges. To avoid liquid moving, the buffer level must be the same in both chambers. A glass plate, laying on the paper bridges, covers the separation plate cooled to 0-4 °C. 

Place the slide into a horizontal electrophoresis apparatus, fill the tanks with Soln. A, and connect the small ends of the slide to the electrode tanks by moistened filter paper wicks. Fill the well directed towards the anode with antiserum dilution and pipet antigen solution containing traces of bromophenol blue in that well which is nearby tbe cathode. 

The species to be compared are loaded in 6% Ficoll (w/v) onto an 8% or 10% polyacrylamide gel with 29 1 monomer bisacrylamide. We generally use 90 mM Tris—borate (pH 8.3) buffer containing either EDTA or added metal salts. Electrophoresis is typically performed for 1—2 days at 5 V/cm at room temperature. When salts are included in the electrophoresis buffer, this must be recirculated between the cathodic and anodic reservoirs at a rate of 1 L/h. This may require a little modification of commercial gel electrophoresis apparatus. Our RNA species generally include at least one strand that is radioactively [5/-32P] -labeled. When the electrophoresis is complete the gel is dried onto Whatman 3MM paper and exposed to storage phosphor plates at 4 °C followed by phosphorimaging to provide a gel image. 

Svensson, H., Physicochemical aspects and their relationship to the design of apparatus. In Paper Electrophoresis, Ciba Foundation Symposium (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 86-104. Churchill, London, 1956. 

Focusing ion exchange". An ingenious application of Inorganic paper electrophoresis has been described in detail by Schumacher in several articles in Helvetica Chimica Acta [40, 221, 228, 234, 2322 (1957) 41, 825, 1771]. He has used the simple apparatus shown in Figs. 15 and 16 to effect separations of carrier-free Sr-Y-Cs in 10 minutes and alternate rare earths in 8 minutes. By adjusting conditions he reports separation of Sr-Y in 5 seconds. 

Place the glass plate with the gel on the electrophoresis apparatus. The connecdon to the buffer in the buffer vessels is made by means of the paper wicks (two layers). 

When the agarose has set, place the plate on the electrophoresis apparatus with the first dimension strip at the cathode end. Connect die gel ith the buffer by means of fresh paper wicks (two layers). 

Place the gel holder in one of the dishes. Put a wetted Scotch-Brite pad on one side of the holder and then three sheets of Whatman paper saturated with transfer buffer on the pad. Place the gel on the paper direcdy after taking it out from the electrophoresis apparatus. The upper stacking gel should be carefully removed because it sticks to nitrocellulose. Pour a few milliliters of transfer buffer on top of the gel and carefully place on it a sheet of prewetted nitrocellulose. Roll over the nitrocellulose with a glass rod to remove any air bubbles between the gel and the membrane. Next, place three Whatman 3MM sheets prewetted with transfer buffer on top of the nitrocellulose, and finally another wet Scotch-Brite pad. 

Merten et al. (M4) precipitated HCl extract of hog gastric mucosa by 60% saturation with ammonium sulfate below pH 6.0 and, after dissolving, submitted it to paper electrophoresis at pH 4.6. The most anodic protein fractions contained proteases with a pepsin to cathepsin ratio of 10.9 1, while the second peak contained both these enzymes at a ratio of 1.6 1. After re-electrophoresis, the first peak demostrated peptic activity only at pH 1.4-2.4, with no activity at pH 3.5. Prolonged electrophoresis in the Tiselius apparatus of crystalline hog pepsin showed heterogeneity of this relatively pure material in acetate and phosphate buffers of pH 3.9, 5.9, and 8.0 (HIO). 

Electrophoresis apparatus and practical considerations The mobility of uridylic acid in 0.05 M ammonium formate buffer pH 3.5 is approximately 0.4 cm hr" volt cm". It is commonly necessary for this nucleotide to run about 40 cm along the paper. So, a potential gradient of 10 volt cm" would be needed to complete the run in 10 hr, i.e. a potential difference of 400 volts. In practice very much larger voltages, up to about 5 kV may be used and separations are then achieved in an hour or so. Currents up to about 10 mA per cm width of paper are used. 

J.3. Experimental procedure for paper electrophoresis The use of a flat-plate water cooled apparatus is described. Brownlee (1972) describes the hanging or up and over types of tank which use an organic coolant. 

The electrophoresis apparatus is prepared by cutting 4 sheets of thick polythene to cover the cooling plates, two sheets below the electrophoresis paper and two above. The electrode vessels are filled with buffer and the wicks prepared. The wicks are 4 small sheets of 3 MM filter cut to bridge the gaps between the electrophoresis paper and the electrode vessels. The papers are soeiked in buffer and washed dialysis membrane is placed over the edges due to contact the electrophoresis paper (Fig. 3.1). 

Fig. 7.9. Flat-gel horizontal electrophoresis apparatus. Design of Vasu (1969), and reproduced here with details supplied by him. The apparatus is made of Perspex and the base plate (1) is 18.6x13.0x0.5 cm. The vertical pieces (2, 3, 4, 5) are all 3 X 1 X 0.2 mm, and the sides are defined by slats, 12 and 13, respectively 10.8 x 0.5 x 0.3 cm and 11.2 x 0.5 x 0.3 cm, and 14 and 16, which are 20 x 1 x 0.2 cm. The glass upper plate, 15, is 16 x 11 x 0.2 cm. This is placed on top of the base plate, resting on the slats (except the side piece 16) mounted as shown, 14 supported by 4 and 5, which are sealed to the plate. Likewise at the ends, are slats 12 and 13, the former supported by 2 and 3. The slot formers stick to the lower plate, and measure 1.2 X 0.2 X 0.2 cm. The acrylamide solution is poured down an inclined glass plate into the gap, and slat 16 is put in place. After polymerisation, the side slats are eased out with a razor blade and the device is inverted. The end pieces are likewise removed, and sheets of Whatman 6 MM paper are used to make contact with the reservoir, one layer above, another below the gel. After loading, and during the run, the gel is covered with a thin plastic sheet.

Another way of dealing with an excised zone is described by Adesnik (1970), and has been adapted to strips of gel cut from broad slabs on which are run as much as 5 mg of RNA (Adams et al. 1969). The gel slice is coarsely chopped up and placed into a cylindrical tube 10 x 1 cm, one end of which is covered with DEAE paper, and over this a layer of cellophane attached to the tube with a rubber band. The tube is filled with buffer (0.04 M Tris acetate, pH 8.3) and placed in a disc electrophoresis apparatus. After electrophoresis the RNA is found trapped in the ion-exchange paper, from which it is eluted as before. 

Vertical gel electrophoresis apparatus, cold room or cold cabinet, Whatman paper sheets, gel dryer, phosphorim-ager screens (GE-Amersham), Phosphorimager (Typhoon or Storm - Molecular Dynamics), 10% (w/v) ammonium persulfate, TEMED and other accessories for normal PAGE experiment as mentioned above. 

Figure 16.2 Diagram of the apparatus used for continuous-flow paper electrophoresis.

---