Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains

Pamela L. Tuma1, Ann L. Hubbard1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.2
DOI:  10.1002/0471143030.cb0302s02
Online Posting Date:  May, 2001
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Abstract

This unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5′ nucleotidase, and K+‐stimulated.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Plasma Membrane Sheets
  • Support Protocol 1: Assay for Alkaline Phosphodiesterase I Activity
  • Basic Protocol 2: Isolation of Plasma Membrane Domains
  • Support Protocol 2: Assay for K+‐Stimulated p‐Nitrophenylphosphatase Activity
  • Support Protocol 3: Assay for 5′‐Nucleotidase Activity
  • Support Protocol 4: Indirect Immunofluorescent Detection of Proteins Associated with Plasma Membrane Sheets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Plasma Membrane Sheets

  Materials
  • 125‐ to 150‐g male Sprague‐Dawley rats
  • Ether
  • 0.9% (w/v) NaCl, ice cold (store 1 to 2 weeks at 4°C)
  • 0.25 and 2.0 M STM solutions (see reciperecipes), ice cold
  • Protease inhibitor solutions (see recipe)
  • 0.25 M sucrose solution (see recipe), ice cold
  • 40‐ml Dounce homogenizer (Wheaton) with tight‐ and loose‐fitting pestles (size A and B, respectively, although certain manufacturers reverse these designations)
  • Cheesecloth, grade 60
  • Abbe refractometer (Bausch and Lomb)
  • 7‐ml Dounce homogenizer (Wheaton) with loose‐fitting pestle (size B)
  • Additional reagents and equipment for SDS‐PAGE unit 6.1), quantitative immunoblotting (unit 6.2), and densitometry (unit 6.3)

Support Protocol 1: Assay for Alkaline Phosphodiesterase I Activity

  Materials
  • Preparative fractions and isolated plasma membrane sheets (see protocol 1)
  • Alkaline phosphodiesterase I reaction buffer (see recipe)
  • 5% and 10% (w/v) trichloroacetic acid (TCA), ice cold
  • 2 N NaOH
  • 0.25 mM p‐nitrophenol in 5% (w/v) TCA
  • 1‐cm‐pathlength cuvettes

Basic Protocol 2: Isolation of Plasma Membrane Domains

  Materials
  • Plasma membrane sheets (see protocol 1)
  • 0.25, 0.46, and 1.42 M sucrose solutions (see reciperecipes)
  • Protease inhibitor solutions (see recipe)
  • Antibodies specific for apical and basolateral domains (e.g., anti–dipeptidyl peptidase IV for apical domain and CE9 antibody for basolateral domain; Hubbard et al., )
  • Sonicating water bath (e.g., Laboratory Supplies)
  • Peristaltic pump
  • Gradient maker
  • Abbe refractometer (Bausch and Lomb)
  • Additional reagents and equipment for preparing and collecting sucrose gradients (unit 5.3), SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and densitometry (unit 6.3)

Support Protocol 2: Assay for K+‐Stimulated p‐Nitrophenylphosphatase Activity

  Materials
  • Domain gradient fractions and resuspended pellet fraction (see protocol 3)
  • p‐Nitrophenylphosphatase reaction buffer, with and without K+ (see recipe)
  • Substrate mix (see recipe)
  • 1 and 10 N NaOH
  • 0.25 mM p‐nitrophenol in 1 N NaOH
  • 1‐cm‐pathlength cuvette

Support Protocol 3: Assay for 5′‐Nucleotidase Activity

  Materials
  • Domain gradient fractions and resuspended pellet fraction (see protocol 3)
  • 5′‐Nucleotidase reaction buffer (see recipe)
  • 5% and 30% (w/v) trichloroacetic acid (TCA), ice cold
  • 10% (w/v) ascorbic acid, prepared fresh
  • 0.42% (w/v) ammonium molybdate (see recipe)
  • 0.1 mM KH 2PO 4 in 10% (w/v) TCA
  • 1‐cm‐pathlength cuvette

Support Protocol 4: Indirect Immunofluorescent Detection of Proteins Associated with Plasma Membrane Sheets

  Materials
  • Plasma membrane sheets (see protocol 1)
  • 0.25 M sucrose solution (see recipe)
  • PBS ( appendix 2A)
  • Methanol, prechilled to −20°C
  • PBS/1% (w/v) BSA, prepared fresh
  • Primary antibody for marker protein
  • PBS/0.2% (w/v) BSA, prepared fresh
  • Fluorochrome‐conjugated secondary antibody specific for Ig of the species of the primary antibody
  • Phenylenediamine mounting medium (see recipe)
  • Nail polish
  • 22 × 22–mm glass coverslips
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Figures

Videos

Literature Cited

Literature Cited
   Bartles, J.R., Braiterman, L.T., and Hubbard, A.L. 1985. Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane. J. Cell Biol. 100:1126‐1138.
   Blouin, A., Bolender, R.P., and Weibel, E.R. 1977. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. J. Cell Biol. 72:441‐455.
   Fujita, H., Tuma, P.L., Finnegan, C.M., Locco, L., and Hubbard, A.L. 1998. Endogenous syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression at the hepatocyte plasma membrane. Biochem. J. 329:527‐538.
   Hubbard, A.L. and Ma, A. 1983. Isolation of rat hepatocyte plasma membranes. II. Identification of membrane‐associated cytoskeletal proteins. J.Cell Biol. 96:230‐239.
   Hubbard, A.L., Wall, D.A., and Ma, A. 1983. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J. Cell Biol. 96:217‐229.
   Hubbard, A.L., Bartles, J.R., and Braiterman, L.T. 1985. Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies. J. Cell Biol. 100:1115‐1125.
   Stieger, B., Marxer, A., and Hauri, H.‐P. 1986. Isolation of brushborder membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme? J. Membr. Biol. 2:19‐31.
   Touster, O., Aronson, N.N., Dulaney, J.T., and Hendrickson, H. 1970. Isolation of rat liver plasma membranes: Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes. J. Cell Biol. 47:604‐618.
   Weibel, E.R. 1976. Stereological approach to the study of cell surface morphometry. Sixth European Congress on Electron Microscopy, Jerusalem, pp. 6‐9.
   Weibel, E.R., Satubli, W., Gnagi, H.R., and Hess, F.A. 1969. Correlated morphometric and biochemical studies of the liver cell. J. Cell Biol. 42:68‐91.
   Widnell, C.C. and Unkeless, J.C. 1968. Partial purification of a lipoprotein with 5′ nucleotidase activity from membranes of rat liver cells. Proc. Natl. Acad. Sci. U.S.A. 61:1050‐1057.
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