FAST ANALYSIS OF PROTEINS BY ISOELECTRIC-FOCUSING PERFORMED IN CAPILLARY ARRAY DETECTED WITH CONCENTRATION GRADIENT IMAGING-SYSTEM
FAST ANALYSIS OF PROTEINS BY ISOELECTRIC-FOCUSING PERFORMED IN CAPILLARY ARRAY DETECTED WITH CONCENTRATION GRADIENT IMAGING-SYSTEM
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DOI：
10.1002/elps.1150140172
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发表时间：
1993-05-01
期刊：
影响因子：
2.9
通讯作者：
PAWLISZYN, J
中科院分区：
文献类型：
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作者：
WU, JQ;PAWLISZYN, J
在 2-4 个短毛细管（4-10 厘米长）阵列中同时进行多个蛋白质样品的等电聚焦。分离的蛋白质通过在线浓度梯度成像检测系统或单点浓度梯度检测器检测，单点浓度梯度检测器需要在聚焦过程之后进行动员步骤。毛细管阵列的应用提高了毛细管等电聚焦（CIEF）技术的通量，使该技术的样品通量与凝胶板等电聚焦技术相当，但分离和定量的速度更快。成像检测系统2-3分钟即可完成分析，比传统凝胶板等电聚焦快约100倍，比传统CIEF快10倍。分离分辨率为 0.02 pH 单位或更好。毛细管阵列中所有分离的蛋白质均由通用浓度梯度检测器检测，无需样品衍生化。这项工作中描述的廉价仪器的检测浓度限约为 30 微克/毫升，可以通过在系统中使用更高质量的组件来降低，例如更稳定的激光器、更高光学质量的毛细管和更快的计算机 A/D 板。对于 4 厘米长、内径 100 微米的方形毛细管，样品消耗量为 400 nL。使用成像检测系统时，在聚焦后，无需内部 pI 标记，即可直接确定样品的等电点 (pI) 值。
Isoelectric focusing of several protein samples is performed simultaneously in an array of 2-4 short capillaries (4-10 cm long). The separated proteins are detected by either an on-line concentration gradient imaging detection system, or a single point concentration gradient detector which requires a mobilization step following the focusing process. The application of the capillary array increases the throughput of the capillary isoelectric focusing (CIEF) technique, and makes the technique comparable in sample throughput to the gel slab isoelectric focusing technique, but with much faster speed of separation and quantitation. The analysis is completed in 2-3 min with the imaging detection system, 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 separated proteins in the capillary array are detected by the universal concentration gradient detector, which eliminates the need for sample derivatization. The concentration limit of detection for the inexpensive instrument described in this work is about 30 mug/mL and can be reduced by using higher quality components in the system, such as a more stable 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 positions inside the capillary after focusing when the imaging detection system is used.