Field-Free Remobilization of Proteins after Isoelectric Focusing in Packed Capillaries
Field-Free Remobilization of Proteins after Isoelectric Focusing in Packed Capillaries
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DOI：
10.1021/ac101680z
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发表时间：
2010-11-01
影响因子：
7.4
通讯作者：
Wirth, Mary J.
中科院分区：
文献类型：
--
作者：
Hua, Yimin;Koshel, Brooke M.;Wirth, Mary J.
压力驱动的再动员没有施加电场被证明是可能的毛细管等电聚焦使用填充毛细管。毛细管尺寸为100 μ m内径。长度为2cm，填料由0.9 μ m的无孔二氧化硅颗粒制成，所述无孔二氧化硅颗粒用聚丙烯酰胺刷层进行化学改性。可逆和不可逆吸附都可以忽略不计。填充的毛细管消除了储液器之间不需要的流体动力学流动的问题。三种蛋白质是重点：胰蛋白酶抑制剂，碳酸酐酶II和肌红蛋白。与开放毛细管相比，在填充毛细管中聚焦所需的时间仅增加2倍，在200 V/cm下在不到15分钟内完成聚焦。填充的毛细管允许使用更高的电场，分辨率持续增加到至少1500 V/cm。场关闭后，填充阻碍扩散增宽：对于胰蛋白酶抑制剂，填充毛细管的D = 6.1（+/- 0.3）x 10（-8）cm（2）/s，而开放毛细管的D = 28.8（+/- 0.3）x 10（-8）cm（2）/s。在再活化过程中由堆积引起的增宽是由涡流扩散引起的，它由其板高H描述，H是单位长度的方差：H = sigma（2）/L = 0.64 μ m。对于pH范围为3-10的2 cm毛细管，这将分离度限制为0.1 pH单位，得到的理论峰容量为47。
Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The capillary dimensions are 100 mu m i.d. and 2 cm in length, and the packing is made of 0.9 mu m nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II, and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of 2 compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D = 6.1(+/- 0.3) x 10(-8) cm(2)/s for the packed capillary vs D = 28.8(+/- 0.3) x 10(-8) cm(2)/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H = sigma(2)/L = 0.64 mu m. This limits the resolution to 0.1 pH units for the 2 cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.