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A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser‐induced fluorescence detection
Author(s) -
Shiraishi Mitsuya,
Loutzenhiser Rodger D.,
Walsh Michael P.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410119
Subject(s) - isoelectric focusing , chemistry , chromatography , phosphorylation , capillary electrophoresis , gel electrophoresis , sodium dodecyl sulfate , fluorescence , myosin , biochemistry , physics , quantum mechanics , enzyme
Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC 20 ) has been implicated in numerous contractile and motile events, e.g. , smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC 20 phosphorylation in minute samples is critical to determine the importance of LC 20 phosphorylation in diverse physiological processes. We have developed a method for the separation and quantification of unphosphorylated, monophosphorylated, and diphosphorylated LC 20 with a detection limit of 1 pg (50 amol). LC 20 is initially isolated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transblotted to a polyvinlyidene difluoride (PVDF) membrane. The region of the membrane containing the LC 20 band (identified by electrophoresis of purified LC 20 in a neighboring lane) is cut out and fluorescently labeled with Alexa Fluor 488 C 5 maleimide. The labeled LC 20 is eluted from the membrane with detergent and subjected to capillary isoelectric focusing (CIEF) to separate unphosphorylated, mono‐, and diphosphorylated LC 20 , which are detected and quantified by laser‐induced fluorescence (LIF). A linear relationship between log(peak area) and log(LC 20 amount) is observed over the range of 50 amol–150 fmol. Quantification of LC 20 phosphorylation by CIEF with LIF detection was compared with three commonly used methods with much lower levels of sensitivity: urea/glycerol‐PAGE with Western blotting, phosphorylation by [γ‐ 32 P]ATP with Čerenkov counting, and phosphorylation by [γ‐ 32 P]ATP followed by SDS‐PAGE, autoradiography, and scanning densitometry. All four methods gave very similar quantitative results, the major difference being that the new method exhibits 〉 3000‐fold enhanced sensitivity. This method is therefore applicable to quantitative analysis of phosphorylation of minute quantities of LC 20 .