Immunofluorescence Analysis of Membrane-Associated Proteins for Clathrin-Mediated Endocytosis in Plant Root Cells

Chao Wang; Xu Yan; Tingting Meng; Tianwei Hu; Jianwei Pan

doi:10.1007/978-1-4939-7262-3_13

Immunofluorescence Analysis of Membrane-Associated Proteins for Clathrin-Mediated Endocytosis in Plant Root Cells

Methods Mol Biol. 2017:1662:151-157. doi: 10.1007/978-1-4939-7262-3_13.

Authors

Chao Wang¹, Xu Yan², Tingting Meng², Tianwei Hu¹, Jianwei Pan³

Affiliations

¹ School of Life Sceicnes, Lanzhou University, 222 South Tianshui Rd, Lanzhou, 730000, China.
² College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, 321004, China.
³ School of Life Sceicnes, Lanzhou University, 222 South Tianshui Rd, Lanzhou, 730000, China. [email protected].

PMID: 28861825
DOI: 10.1007/978-1-4939-7262-3_13

Abstract

The root is an ideal model system for studying subcellular localization and dynamic trafficking of important membrane-associated proteins in plants. Immunofluorescence analysis is necessary to reveal subcellular localization and intracellular trafficking of endogenous proteins as epitope tags or fluorescent proteins may cause mislocation of fusion proteins. Here, we describe a rapid and reliable immunodetection protocol for whole-mount in situ localization of membrane-associated proteins involved in clathrin-mediated endocytosis (CME) in Arabidopsis root cells. The whole procedure includes five basic steps: tissue fixation, tissue permeation, blocking, primary antibody incubation, and secondary antibody incubation.

Keywords: Antibodies; Arabidopsis; Immunolocalization; Membrane-associated proteins; Whole-mount in situ.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Vesicular Transport / genetics*
Adaptor Proteins, Vesicular Transport / metabolism
Antibodies / chemistry
Arabidopsis / drug effects
Arabidopsis / genetics
Arabidopsis / metabolism
Arabidopsis / ultrastructure*
Arabidopsis Proteins / genetics
Arabidopsis Proteins / metabolism
Brefeldin A / pharmacology
Chromosomal Proteins, Non-Histone / genetics
Chromosomal Proteins, Non-Histone / metabolism
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism
Endocytosis / drug effects
Endocytosis / genetics*
Fluorescent Antibody Technique / methods*
Gene Expression Regulation, Plant*
Indoleacetic Acids / pharmacology
MADS Domain Proteins / genetics
MADS Domain Proteins / metabolism
Membrane Transport Proteins / genetics
Membrane Transport Proteins / metabolism
Plant Roots / drug effects
Plant Roots / metabolism
Plant Roots / ultrastructure*
Tissue Fixation / methods

Substances

AP1 protein, Arabidopsis
Adaptor Proteins, Vesicular Transport
Antibodies
Arabidopsis Proteins
BAF60 protein, Arabidopsis
Chromosomal Proteins, Non-Histone
DNA-Binding Proteins
Indoleacetic Acids
MADS Domain Proteins
Membrane Transport Proteins
PIN1 protein, Arabidopsis
PIN2 protein, Arabidopsis
Brefeldin A