Sensitivity Enhancement Methods for Capillary Isoelectric Focusing/ Concentration Gradient Imaging System

Abstract

Proteins in biological cells can be analyzed by the inexpensive capillary isoelectric focusing (CIEF)/concentration gradient imaging system. Proteins were focused during the focusing process by the CIEF instrument which contains a 4-cm-long, 100 μ i. d. capillary. All focused zones were detected in an on-line fashion by the universal concentration gradient imaging detector consisting of a He-Ne laser and charge-coupled device. The sensitivity of the detector was enhanced in two ways; a signal averaging method and an off-column concentration method using ultrafiltration. Sensitivity enhancement by a factor of 4.5 was achieved by the averaging method, and the detection limit was 13 μ/mL. The whole analysis time was only 2 minutes. In the concentration method, protein samples were first concentrated by more than an order of magnitude by using ultrafiltration performed in a centrifuge. Then, they were introduced into the CIEF instrument, and separated and detected. The detection limit was 1 – 5 μg/mL. The whole analysis procedure took only about 25 minutes. This speed is much faster than that of gel slab isoelectric focusing, and while similar to CIEF performed in commercial instruments. The detection limit is much lower than that of commercial CIEF instruments due to the concentration step.