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Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure

Authors

  • Yu-Ki Matsuno,

    1. Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, Tsukuba, Japan
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  • Weijie Dong,

    1. Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, Tsukuba, Japan
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  • Seiya Yokoyama,

    1. Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Suguru Yonezawa,

    1. Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Takuro Saito,

    1. Department of Surgery I, Fukushima Medical University, Fukushima, Japan
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  • Mitsukazu Gotoh,

    1. Department of Surgery I, Fukushima Medical University, Fukushima, Japan
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  • Hisashi Narimatsu,

    1. Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, Tsukuba, Japan
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  • Akihiko Kameyama

    Corresponding author
    1. Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, Tsukuba, Japan
    • Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan Fax: +81-29-861-3123
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  • Colour Online: See the article online to view Fig. 4 in colour.

Abstract

Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME-separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF-II and BxPC3) and also analyzed their O-linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer.

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