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From Cytosol to the Apoplast: The Hygromycin Phosphotransferase (HYGR) Model in Arabidopsis

  • Haiyan Zhang
  • Jinjin Li
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1459)

Abstract

The process by which proteins are secreted via endoplasmic reticulum (ER)/Golgi-independent mechanism is conveniently called unconventional protein secretion. Recent studies have revealed that unconventional protein secretion operates in plants, but little is known about its underlying mechanism and function. This chapter provides methods we have used to analyze unconventional character of hygromycin phosphotransferase (HYGR) secretion in plant cells. Following isolation of protoplasts from HYG R -GFP-transgenic plants and incubation with brefeldin A (BFA), an inhibitor of conventional secretory pathway, we easily obtain protein extracts from protoplasts and culture medium separately. These proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis with anti-GFP antibodies.

Key words

Unconventional protein secretion Hygromycin phosphotransferase Protoplast isolation Trichloroacetic acid precipitation Western blot GFP antibodies 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Key Laboratory of Plant Resources, Institute of BotanyChinese Academy of SciencesBeijingChina

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