Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana

Erica D. LaMontagne1, Carina A. Collins1, Scott C. Peck2, Antje Heese1

1 University of Missouri–Columbia, Division of Biochemistry, Interdisciplinary Plant Group (IPG), Columbia, Missouri, 2 Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20020
Online Posting Date:  May, 2016
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Abstract

Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane‐derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co‐immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc.

Keywords: Arabidopsis thaliana; cellular fractionation; differential centrifugation; microsomes; solubilization of microsomal proteins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Solubilized Microsomal Proteins from Arabidopsis Seedlings
  • Alternate Protocol 1: Isolation of Solubilized Microsomal Proteins from Arabidopsis Suspension‐Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Solubilized Microsomal Proteins from Arabidopsis Seedlings

  Materials
  • 7‐to 8‐day old Arabidopsis thaliana seedlings
  • 0.5× Murashige & Skoog (MS) agar plates (see recipe)
  • Homogenization buffer (see recipe)
  • White quartz sand (Sigma, cat. no. 26‐0010 SAJ)
  • Protein determination assay kit (e.g., Bradford Protein Assay from BioRad)
  • 6× SDS‐PAGE protein sample buffer (Gallagher, )
  • Nonidet P‐40 substitute (Roche, cat. no. 11332473001)
  • Antibodies against subcellular marker proteins for immunoblotting
  • Porcelain mortar with capacity of 50 ml and porcelain pestle with flat bottom
  • Forceps
  • 15‐ml conical tubes
  • Miracloth (Calbiochem, cat. no. 475855‐1R; cut into 8 × 8 cm squares)
  • Microcentrifuge tubes (1.7 ml and 2.0 ml)
  • Tabletop microcentrifuge with temperature control
  • Preparative ultracentrifuge with compatible rotor (e.g., Optima TXL‐120 Preparative Tabletop Ultracentrifuge with TLA 100.2 fixed‐angle rotor)
  • Thick‐walled polycarbonate tubes (7 × 21 mm, Beckman Coulter)
  • Long round‐ended polypropylene pestles [21 cm (length) × 4.8 mm (shaft) conical tissue grinder; Bel‐Art Products, cat. no. F199210001]
  • Spectrophotometer and cuvettes
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and immunoblotting (Gallagher et al., )

Alternate Protocol 1: Isolation of Solubilized Microsomal Proteins from Arabidopsis Suspension‐Cultured Cells

  Material
  • Arabidopsis suspension‐cultured cells (May and Leaver, )
  • 1× Murashige and Skoog (MS) liquid medium (see recipe)
  • Liquid nitrogen
  • White quartz sand (Sigma, cat. no. 26‐0010 SAJ)
  • Homogenization buffer (see recipe)
  • 50‐ml beaker, sterile
  • Rotary shaker
  • Filter paper (Whatman, cat. no. 1002‐070, Grade 2; 70‐mm diameter)
  • Büchner funnel (polypropylene, 80‐mm‐diameter)
  • Vacuum source
  • Side‐arm flask with rubber stopper and rubber tubing
  • Porcelain mortar with capacity of 50 ml and porcelain pestle with flat bottom
  • Metal spatula
  • 15‐ml conical tube
  • Miracloth (Calbiochem, cat. no. 475855‐1R)
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Figures

Videos

Literature Cited

Literature Cited
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