Rapid isoelectric focusing and detection of nanogram amounts of proteins from body tissues and fluids

Abstract Polyacrylamide gel isoelectric focusing was carried out using 250 μm thick gels backed with polyester films at voltage gradients up to 460 V/cm. Peltier cooling was employed with berrylium oxide heat transfer plates to achieve adequate cooling. Visualization of low protein concentrations was obtained by the use of a highly sensitive silver staining technique. Results indicated that over separating distances of 5.4 cm 150 to 200 bands should be resolved and that considerable differences between two commercial carrier ampholytes exist. Pharmalyte carrier ampholytes from Pharmacia have better continuity and conductance over the range of pH 3–10 but require much higher field strengths to achieve maximal separation than do LKB Ampholines over a similar pH range.