SENSITIVITY ENHANCEMENT METHODS FOR CAPILLARY ISOELECTRIC-FOCUSING CONCENTRATION GRADIENT IMAGING-SYSTEM

Proteins in biological cells can be analyzed by the inexpensive capill ary isoelectric focusing (CIEF)/concentration gradient imaging system. Proteins were focused during the focusing process by the CIEF instrum ent which contains a 4-cm-long, 100 mum i.d. capillary. All focused zo nes were detected in an on-line fashion by the universal concentration gradient imaging detector consisting of a He-Ne laser and charge-coup led 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 achie ved by the averaging method, and the detection limit was 13 mug/mL. Th e whole analysis time was only 2 minutes. In the concentration method, protein samples were first concentrated by more than an order of magn itude by using ultrafiltration performed in a centrifuge. Then, they w ere introduced into the CIEF instrument, and separated and detected. T he detection limit was 1-5 mug/mL. The whole analysis procedure took o nly about 25 minutes. This speed is much faster than that of gel slab isoelectric focusing, and while similar to CIEF performed in commercia l instruments. The detection limit is much lower than that of commerci al CIEF instruments due to the concentration step.