Detection of Nε-(hexanoyl)lysine in the tropomyosin 1 protein in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric cancer cells

Journal of Clinical Biochemistry and Nutrition (2011)

Hitomi Okada Yuji Naito Tomohisa Takagi Megumi Takaoka Tomoko Oya‐Ito Kohei Fukumoto Kazuhiko Uchiyama Osamu Handa Satoshi Kokura Yumiko Nagano Hirofumi Matsui Yoji Kato Toshihiko Osawa Toshikazu Yoshikawa

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Abstract:

Nε-(Hexanoyl)lysine, formed by the reaction of lysine with n-6 lipid hydroperoxide, is a lipid peroxidation marker during the initial stage of oxidative stress. The aim of the present study is to indentify Nε-(hexanoyl)lysine-modified proteins in neoplastic transformed gastric mucosal cells by N-methyl-N’-nitro-N-nitrosoguanidine, and to compare the levels of these proteins between gastric mucosal cells and normal gastric cells. Much greater fluorescence of 2-[6-(4'-hydroxy)phenoxyl-3H-xanthen-3-on-9-yl]benzoic acid, an index of the intracellular levels of reactive oxygen species, was observed for gastric mucosal cells compared to normal gastric cells. Nε-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-Nε-(hexanoyl)lysine polyclonal antibody, and a protein band of between 30–40 kDa was clearly increased in gastric mucosal cells compared to normal gastric cells. Two Nε-(hexanoyl)lysine-modified protein spots in gastric mucosal cells were identified as the tropomyosin 1 protein by mass spectrometry using a MASCOT search. The existence of Nε-(hexanoyl)lysine modification in tropomyosin 1 was confirmed by Western blotting of SDS-PAGE-separated or two-dimensional electrophoresis-separated proteins as well as by the immunoprecipitation with anti-tropomyosin 1 antibody. These data indicate that Nε-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine in gastric epithelial cells.

Keywords:

Tropomyosin

Polyclonal antibodies

Topics:

Electron Spin Resonance Studies

Metabolomics and Mass Spectrometry Studies

Redox biology and oxidative stress

10.3164/jcbn.11-39

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Microchamber Western Blotting Using Poly-l-Lysine Conjugated Polyacrylamide Gel for Blotting of Sodium Dodecyl Sulfate Coated Proteins

Analytical Chemistry (2013)

Minsub Chung Do-Hyun Kim Amy E. Herr

We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

Sodium dodecyl sulfate

Polyacrylamide

10.1021/ac401012j

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Citations (12)

SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting

Methods in molecular biology (2018)

Edward P. Trieu Ira N. Targoff

Immunoprecipitation

Sodium dodecyl sulfate

10.1007/978-1-4939-8793-1_35

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