Electrophoresis constant current

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature.

Regulated direct current (DC) power supplies designed for electrophoresis allow control of every electrophoretic mode. Constant voltage, constant current, or constant power conditions can be selected. Many power supplies have timers and some have integrators allowing runs to be automatically terminated after a set time or number of volt-hours (important in IEF). All modes of operation can produce satisfactory results, but for best results and good reproducibility some form of electrical control is important. The choice of which electrical parameter to control is almost a matter of preference. The major limitation is the ability of the chamber to dissipate the heat generated by the electrical current. 

The electrophoresis is controlled by a power supply mostly in the constant current (cc) or constant voltage (cv) mode. It is advised against application of constant current during separation, because during the run the electric resistance increases, and because current is constant, voltage and heat production increase, too. 

Prior to protein separation, a pre-electrophoresis is essential. It is done overnight with 3.5 mA/cm (constant current). The electrode buffer D is renewed after pre-electrophoresis. 

Under this condition of reduced solution amounts, the amount of the SDS ions in the electrolyte solution decreases with time and protein mobility during electrophoresis becomes gradually reduced if the applied potential is maintained at a constant voltage. Applying the condition of constant current, the voltage is increased to maintain the separation mobility in the latter half of the process. 

Figure 8.1—Principle of zone electrophoresis. Each compartment is separated by a membrane to avoid contamination of the electrolyte by secondary products formed at the electrodes. The size and the sign of the charge carried by each species depends on the chemical medium in which they are found. The experiment can be carried out at constant current, constant voltage or constant power.

The procedure used for the gels shown in Fig. 4.2 was that of Swank and Munkres (ref. 328), except the samples were run for 18 hr at a constant current of 1.5 mA/gel and the protein-fixing solution used after electrophoresis contained 4 5 1 methanol-water-trichloroacetic acid. 

Fig. 56. Star electrophoresis. Buffer, Veronal-sodium Veronalate, pH 8.6 p 0.06 substrate, Whatman 3 MM, dimensions 22 x 30 cm electrical field constant current 26 ma starting point, 17 volts/cm endpoint, 11 volts/cm sample, 0.02 ml normal human serum duration, 3 hours.

Polyacrylamide gel electrophoresis is conducted utilizing a published procedure [32], Samples of approximately 50 //g of the antibody solution were subjected to electrophoresis. The buffer solution was of pH 8.3 and consisted of 0.005 M Tris and 0.04 M glycine. Electrophoresis was conducted at a constant current of 2.5 ma/gel for periods of 4 to 6 hrs. The finished gels were stained with 0.02% Coomassie Blue G-250 to reveal the protein components. The results for immune serum and the purified antiglucose antibodies were photographed, Fig. (8A). The non-antibody protein components in the serum have been removed by the affinity method. 

Fig. 3.10. Autoradiograph of a sequencing gel prepared using the Maat and Smith procedure. The sequence shown is that derived from a 440 nucleotide-long fragment from a Hinfl digest of a 5 -end labelled HirtdUl fragment of adenovirus type 5 DNA. Samples from each base-specific reaction mixture were loaded every 2 hours (runs I, II, III, and IV). Electrophoresis was carried out at a constant current of 30 mA. Nucleotide sequence analysis of the complementary DNA strand revealed one mistake in the sequence as written. At position 2870 (in run III) two C s should be read instead of one. The zone of compression responsible for this error is not very apparent and emphasizes the importance of sequencing both DNA strands.

Power supply for high-voltage electrophoresis LKB Biochrom 2103 power supply unit, LKB Instruments Ltd., 232 Addington Road, Selsdon, South Croydon, Surrey CR2 8YD, U.K. This instrument may be used to run gels at constant voltage, constant current or constant power. 

Perform the electrophoresis at 50 mA, constant current. You will see two dye fronts develop one from the bromophenol blue dye (dark blue) and one from the xylene cyanole dye (light blue). The latter of the two dyes will migrate the same as a DNA fragment of approximately 4 kb, while the former dye will migrate the same as a DNA fragment of about 0.5 kb. 

Apply 50 mA (constant current) and continue the electrophoresis until the bromophenol blue (dark blue) tracking dye has migrated about three-fourths of the way to the end of the gel. 

Mobility, Determination by Zone Electrophoresis at Constant Current... 

Zone Electrophoresis, at Constant Current, Mobility Determination by... 

Carry out electrophoresis at 50-60 mA constant current/gel until the sample has traversed the stacking gel during this time, the samples stack, becoming concentrated into a very tight band, affording maximal resolution and separadon of the polypeptides in the separating gel. Then continue electrophoresis at 25-30 mA/gel until the bromophenol blue dye front is approx 1-2 cm from the end of the gel Note 4). 

Electrophoresis The buffer reservoirs are filled with cold (about 5°C) electrode buffer and any bubbles trapped at the top or bottom of the gel are removed. Electrophoresis is carried out at a constant current of 4 mA/tube (applied voltage about 90 V at the outset). Electrophoresis is continued until the bromophenol blue band migrates to about 1 cm from the bottom of the tube, usually about 70 min. 

Figure 9.10. Voltage versus distance for (a) a linear electrophoresis gel, and (b) a gradient gel. From Ohm s law, with constant current, the observed voltage changes mirror the resistance properties of the gels.

For isoelectric focusing (IFF) (see later section), a power supply that provides constant power is advisable. During electrophoresis, current drops significantly because of lower conductivity as carrier ampholytes focus at their isoelectric points and because of creation of zones of pure water. If a constant-voltage supply is used, frequent voltage adjustments may be necessary. Constant-current power supplies are not customarily used in lEF. Pulsed-power or pulsed-field techniques (see later section) require a power supply that can periodically change the orientation of the applied field relative to the direction of migration. 

Figure 11. Separation of treated and untreated human erythrocytes by low-electric-field electrophoresis. Human erythrocytes (5 x 107) treated with neuraminidase are mixed with 5 x 107 untreated erythrocytes to a final volume of 5 ml and layered onto a linear gradient of 1.5-cm height. Electrophoresis proceeds for 25 min at 5°C at a constant current of 90 mA. An identical suspension is subjected to velocity sedimentation at unit gravity only. (O) Separation at unit gravity ( ) separation at unit gravity and by electrophoresis. Areas under the migration profile are depicted in shading. Gravity and electric forces act in the same direction (to the right). (Figures 8—11 reproduced with kind permission of the editor of Anal. Biochem.)...

Electrophoresis Zone, Constant Current Mobility Determination by (Waldmann-... 

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