Silver staining of an esterase compatible with activity and mass spectrometry analysis after separation using non-denaturing two-dimensional electrophoresis

• Published in: Clinica chimica acta Vol. 390; no. 1; pp. 145 - 147
• Main Authors: Shimazaki, Youji, Watanabe, Sono
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2008
• Subjects: 2-DE; Animals; Electrophoresis, Gel, Two-Dimensional - methods; Enzyme activity; Esterases - metabolism; High density lipoprotein; Lipoproteins, HDL - blood; Liver - enzymology; Mass spectrometry; Mass Spectrometry - methods; Mice; Nanotechnology; Silver stain; Silver Staining; HDL; Enzyme activity; High density lipoprotein; ESI; 2-DE; IEF; MS/MS; PVDF; Silver stain; Mass spectrometry
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2007.12.017

Enzyme activity is normally lost when formaldehyde is used as a reductant for silver staining after separation by electrophoresis. Hydrolytic activity of esterases can be examined on membranes without impairing enzyme activity when another reductant such as glucose is used for silver staining of the enzyme after separation by non-denaturing two-dimensional electrophoresis (2-DE) and subsequent transfer. The hydrolysis of lipids in human high density lipoprotein (HDL) by esterases first separated on a polyvinylidene fluoride membrane using non-denaturing 2-DE and silver stained using glucose as a reductant was examined. Esterase activity was retained after glucose was used as a silver reductant for silver staining after separation using non-denaturing 2-DE. Lipids of HDL were removed by the esterases retained on the membrane after esterases were separated by 2-DE. The results indicated that hydrolytic enzyme activity is retained after separation, staining and immobilization.