Nonequilibrium pH Gel Electrophoresis (NEPHGE)

Nonequilibrium pH gel electrophoresis (NEpHGE) is a technique developed to resolve proteins with extremely basic isoelectric points (pH 7.5–11.0) (1 ,2 ). These proteins are difficult to resolve using standard IEF, because the presence of urea in IEF gels has a buffering effect and prevents the pH gradient from reaching the very basic values (with a pH above 7.3–7.6) (3 ). In addition, cathodic drift causes many very basic proteins to run off the end of the gel. During NEPHGE, proteins are not focused to their isoelectric point, but instead move at different rates across the gel owing to charge. For this reason, the accumulated volt hours actually determine the pattern spread across the gel. It is therefore imperative that volt hours be consistent to assure reproducible patterns.