Citation: GAO Peifeng, ZHAO Xinying, HE Muyi, LIU Qingsheng, QU Feng. Comparison of one-step and two-step methods for pI determination of proteins and polypeptides by capillary isoelectric focusing[J]. Chinese Journal of Chromatography, ;2013, 31(6): 537-542. doi: 10.3724/SP.J.1123.2013.03059 shu

Comparison of one-step and two-step methods for pI determination of proteins and polypeptides by capillary isoelectric focusing

GAO Peifeng ¹ ,
ZHAO Xinying ^1,2 ,
HE Muyi ¹ ,
LIU Qingsheng ³ ,
QU Feng ^1,,

1.
1. School of Life Science, Beijing Institute of Technology, Beijing 100081, China;

Corresponding author: QU Feng,
Received Date: 31 March 2013
Available Online: 25 April 2013
Fund Project: 国家自然科学基金项目(21175011,20875009) (21175011,20875009)科技部基础研究计划项目(2012CB910603) (2012CB910603)"十二五"农村领域国家科技计划项目(2011BAD26B040406). (2011BAD26B040406)

One-step and two-step capillary isoelectric focusing (cIEF) methods were employed for the separation and pI determination of proteins and polypeptides. The parameters affecting the analysis efficiency, such as the sample solution, injection volume, focusing voltage, focusing time and driving conditions were optimized. The comparison of the two methods for separation of cytochrome C, hemoglobin, myoglobin, transferrin, bovine serum albumin and six polypeptides showed that the one-step cIEF was simple and fast, which could determine the pI of single component as well as it was rapid for protein and polypeptide separation, but it could not get good resolution or accurate pI of each component in a mixed sample. The two-step cIEF was more complex and needed longer time, however, which could separate and exactly determine the pI of each component in the mixture, and the pI value of each component determined was consistent with that determined using a single sample. The two methods are complementary, and can be widely used in rapid and accurate determination of the pI of amphiphilic biological particles.
- capillary isoelectric focusing,
- one-step,
- two-step,
- protein,
- polypeptide
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