Automated gel electrophoresis: other methods

Isoelectric focusing: For separations according to differences in isoelectric point (pI), PhastGel IEF media provide stable, linear pH gradients (3–9, 4–6.5 and 5–8) with uniform conductivity across broad and narrow pH ranges. PhastGel IEF media withstand field strengths >500 V/cm for high resolution separations. PhastGel Dry IEF is supplied precast and dried. This gel contains no residual acrylamide monomers or other contaminants from the polymerization process, thereby offering greater versatility and reliable band separation. Before use, the gel is reswollen with the chosen carrier ampholyte solution and other additives as required for the application. Reswelling takes place within a PhastGel Cassette to ensure controlled, reproducible rehydration. The process takes from 0.5–2 h depending on the composition of the rehydration solution. Guidance for choosing rehydration solutions is supplied with the product. A dedicated PhastGel kit is available for the detection of oligoclonal IgG in unconcentrated cerebrospinal fluid, the CSF Analysis Kit includes gels and reagents for 40 analyses. Development processes for detection of bands are similar to native and SDS-PAGE gels.

Simple, controlled separations: Programming: Up to nine separation methods and nine development methods can be programmed directly and stored within the PhastSystem separation and control unit. Help messages appear on screen to assist with programming or warn of programming errors. Methods can be named, edited, and programmed while the system is running. A battery back-up ensures that no methods are lost in the event of a power failure and a diagnostic check, performed each time the unit is switched on, warns of any technical problems. The actual separation or staining conditions can be monitored in real time throughout the experiment.

Separation methods:  Each separation method contains instructions to lower and raise the sample applicators, an extra alarm instruction and up to nine other steps that are used to specify run conditions at the programmed time.

Development methods: Each development method can contain up to 20 steps, allowing full flexibility for optimization of staining and destaining protocols.

Easy set up of gel and buffers: PhastGel media are precast and ready to use eliminating time-consuming and often unreliable gel casting. Agarose based PhastGel Buffer Strips eliminate buffer preparation steps. The electrode solutions for native, SDS-PAGE or DNA separations are contained in the strips which are placed on both ends of the gel. The electrodes rest on the surface of the strips. No electrode solutions are required for IEF and the electrodes rest directly on the gel surface.

Accurate sample loading and application: A sample applicator is selected according to the number of samples and the volume that is to be loaded on the gel. The comb-like applicators are available in 12 × 0.3 µl, 8 × 0.5 µl, 8 × 1 µl and 6 × 4 µl formats. Samples are loaded simply by dipping the comb into sample droplets held in place on a sample-well stamp. The capillary wells of the PhastGel sample applicators hold samples until the applicator is lowered onto the gel after the correct, preprogrammed period of pre-electrophoresis. The applicators are then raised to avoid the risk of band disturbances.

Ensuring high resolution and reproducibility: Up to two gels can be run simultaneously under the same conditions within the separation unit. To ensure high resolution and high reproducibility, conditions during the electrophoretic separation are precisely controlled.

Accurate temperature control: The high field strengths required for fast, high resolution separation can generate significant increases in temperature, but constant temperature is essential for reproducible results. The programmable temperature range extends from 0 –70 °C. However, the cooling capacity of the separation bed depends on the ambient temperature, humidity, and the power applied to the gels. The temperature of the separation bed is therefore measured at one second intervals and fed back to an in-built Peltier element that cools or warms the bed, regulating the running temperature. For standard runs, the bed can be cooled to approximately 15 °C below ambient temperature and held to within ±1 °C.  

Accurate power supply: The power supply in the separation and control unit operates in one of three modes: constant current, constant voltage, or constant power. Limits on these parameters are set when programming the method. PhastSystem automatically adjusts the parameters during a run. The power supply accurately delivers voltage and current even at low field strengths (10V, 0.1 mA). Up to 540 V/cm can be applied to the gels, ideal for high resolution IEF.

Volthour control: For maximum reproducibility, PhastSystem uses volthours, to regulate the duration of separation and the period for sample application. A volthour integrator continuously integrates the voltage applied with respect to time controlling protein migration in the gel.