FAST ANALYSIS OF PROTEINS BY ISOELECTRIC-FOCUSING PERFORMED IN CAPILLARY ARRAY DETECTED WITH CONCENTRATION GRADIENT IMAGING-SYSTEM

Isoelectric focusing of several protein samples is performed simultane ously in an array of 2-4 short capillaries (4-10 cm long). The separat ed proteins are detected by either an on-line concentration gradient i maging detection system, or a single point concentration gradient dete ctor which requires a mobilization step following the focusing process . The application of the capillary array increases the throughput of t he capillary isoelectric focusing (CIEF) technique, and makes the tech nique comparable in sample throughput to the gel slab isoelectric focu sing technique, but with much faster speed of separation and quantitat ion. The analysis is completed in 2-3 min with the imaging detection s ystem, which is about 100 times faster than the conventional gel slab isoelectric focusing and 10 times faster compared to conventional CIEF . The resolution of the separation is 0.02 pH units or better. All sep arated proteins in the capillary array are detected by the universal c oncentration gradient detector, which eliminates the need for sample d erivatization. The concentration limit of detection for the inexpensiv e instrument described in this work is about 30 mug/mL and can be redu ced by using higher quality components in the system, such as a more s table laser, higher optical quality capillaries and a faster computer A/D board. The sample consumption is 400 nL for the 4 cm long, 100 mum ID square capillary. The isoelectric point (pI) values of the samples can be directly determined without internal pI markers from their pos itions inside the capillary after focusing when the imaging detection system is used.