Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Abstract

AbstractThree common alleles are found in esterase D: EsD 1, EsD 2 and EsD 5. In addition, a number of rare variants are found. The isoeiectric points of the EsD 1 and EsD 2 isoenzymes differ by only 0.02 pH units, which could lead to possible confusion between the two using conventional isoelectric focusing (IEF). Therefore, in order to ensure adequate separation of these two bands, it is necessary to use a narrow pH gradient. The separation of esterase D (EsD) has been improved on electrofocusing gels by incorporating a narrow range Pharmalyte (pH 4.5–5.4) in conjunction with the separator N‐(2‐hydroxyethyl)piperazine‐N′‐2‐ethanesulphonic acid (HEPES). This mixture resulted in the formation of a narrow pH range of pH 4.85–5.35 (the major typing bands of EsD fall within the range of pI 4.9–5.2). Resolution was further improved by the use of thickness modified pH gradients in ultrathin gels which flattened the pH gradient in the range of pH 5.0–5.1. The techniques used were shown to be effective for typing bloodstains up to 1 month old.