A simple, rapid and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence
A simple, rapid and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence
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DOI：
10.1002/elps.200305428
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发表时间：
2003-06-01
期刊：
影响因子：
2.9
通讯作者：
Chang, HT
中科院分区：
文献类型：
--
作者：
Chiu, TC;Lin, YW;Chang, HT
本文描述了聚环氧乙烷（PEO）电渗逆流毛细管电泳-激光诱导荧光（CE-LIF）分段填充法分析SYPRO Red标记的十二烷基硫酸钠（SDS）-蛋白质（SRSPs）。结果表明，SDS和盐在决定SRSP的荧光强度方面起着至关重要的作用。虽然荧光测量表明，SRSPs在含有0.1%SIDS和高浓度NaCl（100 mM）的Tris-硼酸盐（TB）缓冲液中发出强烈荧光，但鉴于焦耳加热，这些条件不适合CE。为了克服该障碍，我们在注射在含有50 mM NaCl和SYPRO Red的TB缓冲液中制备的样品（0.64穆尔）之前，应用0.1%SIDS的塞（注射体积的1/5至1/3）。当使用背景电解质的0.6%PEO在TB缓冲液中含有NaCl，电渗逆流的分析物允许一个浓缩大的样品体积（高达1/3的有效毛细管长度）在21分钟内，检测0.35和0.10 nm的牛血清白蛋白和酪蛋白，分别。线性动态范围从10 nm到5 μ M，该方法提供了测定牛奶中酪蛋白浓度为0.45 +/- 0.03 mm（n = 5）的能力。
We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP. Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1% SIDS and high concentrations of NaCl (100 mm), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SIDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 muL) prepared in TB buffer containing 50 mm NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nm for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nm to 5 muM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mm (n = 5).