Electrophoresis constant voltage

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. 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

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. 

Electrophoresis is run for about 18 h at RT with constant voltage of 25-30 V. Electrode buffer is Soln. A, which circulates between the electrode chambers during 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.

Run gels at constant voltage, 25-30 mA per slab gel, until the bromophenol blue marker dye has readied the bottom of the gel (about 1 hour). When completed, turn off and unplug the electrophoresis apparatus and remove the gel. Carefully cut the gel between lanes 5 and 6 so that you have two identical halves. Cut a notch in the corner of each gel half to indicate proper orientation. The half containing lanes 1-5 will be stained with Coomassie Blue to detect the location of all proteins. The other half (lanes 6-10) will be blotted onto a nitrocellulose membrane to analyze for glycoproteins. 

Three simple, on-line radioisotope detectors for capillary electrophoresis were described and characterized for the analysis of 43P-labeled analytes. The minimum limit of detection for these systems was shown to be strongly dependent upon the conditions under which the analysis is performed. For standard CE separations performed at a relatively high (constant) voltage, the minimum limit of detection was found to be in the low nanocurie (injected sample... 

Electrophoresis procedure The electrophoresis is run at 200 V with a constant voltage at the room temperature. When the BPB is reached a point of 3 mm from the bottom of the separating gel, the electrophoresis is terminated. Strip the gel from the plate and stain. 

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. 

Load the samples on a 10-well, 12% SDS polyacrylamide gel with a 4% polyacrylamide stacking gel. Two groups can share a single SDS-PAGE gel. Apply 200 V (constant voltage), and allow the electrophoresis to continue until the bromophenol blue dye front reaches the bottom of the gel. 

Reconnect the power supply and apply 200 V (constant voltage). Continue the electrophoresis until the bromophenol blue (dark blue) dye just migrates off of the bottom of the gel into the lower buffer chamber. Any unincorporated radiolabeled nucleotide that was present in the samples will run off of the bottom of the gel with the dye. Remember that the buffer in the lower chamber is now radioactive. 

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. 

Pour gel and allow gel to solidify. After the gel is set, submerge with 1 x running buffer and prerun gel for 5 min at constant voltage (rapid electrophoresis 5 V/cm or for most systems a setting of (6.25 X gel length) V) and run sample. Samples may be applied in duplicate so that half the gel can be stained and the other half can be used for hybridization. Recirculation is not necessary, but buffer may be remixed after 2 h. The run can be stopped when the bromophenol blue is about half way through the gel. 

Run gels at constant voltage, 23-30 mA per slab gel, until the bromophenol blue marker dye has reached the bottom of the gel (about 1 hour). When completed, turn off am the electrophoresis apparatus and remove the gel... 

FIGURE 17.7 Computer-simulated distributions of the 140 carrier components and the three dyes for the pH 5-8.5 gradient system after 10, 1000, and 10,000 min of constant voltage application. The numbers refer to the pi values of the dyes and the arrowheads mark their locations. Successive graphs are presented with a y-axis offset of 30 mM. The insets a and b depict the concentration profiles of the pi 6.6 and 7.4 amphoteric dyes and the pH profiles, respectively, at the indicated time points. Simulations were performed with Ax = 50 xm and having column ends that are impermeable to any sample and carrier compounds. (Modified from Mosher, R.A. and Thormann, W., Electrophoresis, 23, 1803, 2002. With permission.)... 

The reproducible analysis of peptides was one of the first successes of capillary electrophoresis. Acidic peptides, that have proved difficult to separate by LC, are readily separated in low pH buffer systems, such as 100 mM sodium phosphate at pH 2.5. In figure 13.6A, the separation of 10 peptides has been achieved with baseline separation in 15 minutes. Each peptide was injected at a concentration 50 ng/ml in 5 mM phosphate, pH 2.5. The sample was introduced by an 8 s injection, at 8 kV, into a 27 cm x 50 pm capillary, equilibrated with 50 mM phosphate buffer and separated with a constant voltage of 10 kV at 28°C. The solutes were detected by UV adsorption at 200 nm. 

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