Isolation and Use of Rafts

Deborah A. Brown1

1 State University of New York at Stony Brook, Stony Brook, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.10
DOI:  10.1002/0471142735.im1110s51
Online Posting Date:  November, 2002
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Abstract

This unit describes methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft‐like membranes isolated by other methods, they are referred to here as detergent‐resistant membranes (DRMs). DRMs can be isolated by flotation on sucrose density gradients or by pelleting after detergent extraction. DRM proteins can be analyzed by SDS‐PAGE and immunoblotting. Additionally, radiolabeled DRM proteins can be analyzed, and lipids can be quantitated by high‐performance thin layer chromatography. Support protocols needed for the lipid analysis are also provided. Finally, protocols for raft disruption by cholesterol removal and measuring the kinetics of such removal are included together with a method that reverses the cholesterol removal (cholesterol repletion).

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Detergent‐Resistant Membranes by Sucrose Gradient Flotation and Analysis of Proteins by Immunoblotting
  • Alternate Protocol 1: Preparation of Detergent‐Resistant Membranes by Centrifugation
  • Support Protocol 1: Pulse‐Chase Analysis of Detergent‐Resistant Membrane Proteins
  • Support Protocol 2: Detecting the Total Profile of Detergent‐Resistant Membrane Proteins by Radioactive Labeling
  • Support Protocol 3: Analysis of DRM Lipids by Quantitative High‐Performance Thin‐Layer Chromatography (HP‐TLC)
  • Support Protocol 4: Preparation of DEAE Sephadex
  • Support Protocol 5: Preparation of the C18 Reversed‐Phase Column
  • Support Protocol 6: Disruption of Rafts by Cholesterol Removal with Methyl‐β‐Cyclodextrin
  • Support Protocol 7: Determination of Kinetics of Cholesterol Removal Following Methyl‐β‐Cyclodextrin (MBCD) Treatment
  • Support Protocol 8: Cholesterol Repletion of Cells Using MBCD‐Cholesterol Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Detergent‐Resistant Membranes by Sucrose Gradient Flotation and Analysis of Proteins by Immunoblotting

  Materials
  • Cells, adherent or nonadherent
  • recipeTNE buffer (see recipe)
  • recipeTNE/P buffer (see recipe), alone or containing one or more of the following:
  •  1%, 2%, or 5% (v/v) Triton X‐100
  •  0.1 M sodium carbonate
  •  5%, 35%, or 80% (w/v) sucrose
  •  60 mM octyl glucoside
  • Detergent‐compatible protein assay kit (e.g., Bio‐Rad)
  • or 6× SDS/sample buffer (unit 8.4)
  • Acetone (optional), ice cold
  • Diethyl ether (optional)
  • 10‐cm tissue culture dishes (for adherent cells)
  • 1‐ and 5‐ml syringes
  • 22‐G needles
  • SW41 rotor and ultracentrifuge tubes (Beckman)
  • Additional reagents and equipment for immunoprecipitation (unit 8.3), SDS‐PAGE (unit 8.4), and immunoblotting (unit 8.10)

Alternate Protocol 1: Preparation of Detergent‐Resistant Membranes by Centrifugation

  • recipePellet lysis buffer (see recipe)
  • 20% (w/v) SDS ( appendix 2A)
  • 35‐mm tissue culture dishes (for adherent cells)

Support Protocol 1: Pulse‐Chase Analysis of Detergent‐Resistant Membrane Proteins

  • Methionine‐free medium containing recipedialyzed serum (see recipe for dialyzed serum; use amount appropriate for cells)
  • [35S]Methionine
  • Unlabeled methionine
  • Additional reagents and equipment for autoradiography ( appendix 3J)
CAUTION: Volatile 35S‐containing compounds can be released during the labeling procedure (refer to Safety Precautions for Radioisotopes in appendix 1Q).

Support Protocol 2: Detecting the Total Profile of Detergent‐Resistant Membrane Proteins by Radioactive Labeling

  • PBS ( appendix 2A), sterile
  • Methionine‐free growth medium with recipedialyzed serum (see recipe for dialyzed serum; use amount appropriate for cells)
  • Normal growth medium with recipedialyzed serum (see recipe for dialyzed serum)
  • [35S]Methionine
  • Additional reagents and equipment for autoradiography ( appendix 3J)
CAUTION: Volatile 35S‐containing compounds can be released during the labeling procedure (refer to Safety Precautions for Radioisotopes in appendix 1Q).

Support Protocol 3: Analysis of DRM Lipids by Quantitative High‐Performance Thin‐Layer Chromatography (HP‐TLC)

  Materials
  • Methanol
  • Chloroform
  • Solvent A: 30:60:8 (v/v/v) chloroform/methanol/water
  • Siliconizing reagent (Sigmacote, Sigma)
  • DEAE Sephadex slurry (see protocol 6)
  • Solvent B: 30:60:8 (v/v/v) chloroform/methanol/0.8 M sodium acetate
  • 0.1 M NaCl
  • Solvent C: 3:48:47 (v/v/v) chloroform/methanol/0.1 M NaCl
  • Solvent D: 3:48:47 (v/v/v) chloroform/methanol/water
  • Nitrogen gas
  • recipeNeutral lipid standards (see recipe)
  • recipeAcidic lipid standards (see recipe)
  • Acetic acid
  • Formic acid
  • Hexane
  • Isopropyl ether
  • recipeLipid visualization reagent (see recipe)
  • SW28 rotor and ultracentrifuge tubes (Beckman)
  • 15‐ml glass tubes with Teflon‐lined caps
  • High‐powered bath sonicator (Laboratory Supplies, model G112SP1T)
  • 24‐mm Whatman GF/C glass‐fiber filters
  • Filtration apparatus: glass microanalysis filter holder assembly (Fisher) or 24‐mm Büchner funnel with rubber collar inserted in 125‐ml side‐arm flask
  • 125‐ml flat‐bottom flasks with ground‐glass stoppers
  • Glass wool
  • 10‐ml wide‐neck glass pipet, disposable
  • Glass rod
  • 125‐ml flat‐bottom tubes with ground‐glass neck
  • Rotary evaporator
  • 5‐ml conical glass tubes with Teflon‐lined caps
  • Reversed‐phase C18 column (see protocol 7)
  • 125‐ml side‐arm flask with one‐hole stopper
  • 10 × 20–cm high‐performance thin‐layer chromatography (HP‐TLC) plate without fluorescent indicator (Merck)
  • TLC tanks with glass covers
  • Ovens preheated to 100°, 110°, and 180°C
  • Modified 10‐µl Hamilton syringe: syringe with beveled edge ground down to decrease the angle by half, facilitating loading of small drops
  • 100‐ml graduated cylinders with ground‐glass stoppers
  • Blotting paper cut to fit tanks
  • Hair dryer with cool setting (optional)
  • 0.5‐ to 1‐cm thick aluminum block
  • Densitometer
  • Additional reagents and equipment for preparation of DRMs (see protocol 1)
NOTE: All glassware that will touch DEAE Sephadex should be siliconized before use. The column and glass wool are siliconized together after the glass wool is in place.

Support Protocol 4: Preparation of DEAE Sephadex

  Materials
  • DEAE Sephadex A‐25
  • 1 M NaOH
  • 0.5 M acetic acid (57.5 ml glacial acetic acid diluted to 2 liters with water)
  • Methanol
  • Solvent A: 30:60:8 (v/v/v) chloroform/methanol/water
  • 4‐liter side‐arm flask
  • 18.5‐cm‐diameter Büchner funnel with filter paper
  • 250‐ml reagent bottle with ground‐glass stopper or Teflon‐lined screw cap
NOTE: All glassware that will touch DEAE Sephadex should be siliconized before use by rinsing briefly with siliconizing reagent (Sigmacote; Sigma).

Support Protocol 5: Preparation of the C18 Reversed‐Phase Column

  Materials
  • Reversed‐phase C18 granular silica (Millipore)
  • Chloroform
  • Methanol
  • Glass wool
  • 5‐ml disposable glass pipet
  • 125‐ml side‐arm flask with one‐hole stopper
  • 1‐ml disposable glass pack

Support Protocol 6: Disruption of Rafts by Cholesterol Removal with Methyl‐β‐Cyclodextrin

  • PBS ( appendix 2A)
  • Growth media lacking serum, prewarmed
  • Methyl‐β‐cyclodextrin (MBCD; Sigma)
  • Bovine serum albumin (BSA)
  • recipeBuffered saline solution (BSS; see recipe)

Support Protocol 7: Determination of Kinetics of Cholesterol Removal Following Methyl‐β‐Cyclodextrin (MBCD) Treatment

  Materials
  • 60 to 100 Ci/mmol [1,2,6,7‐3H(N)]‐cholesterol ([3H]cholesterol)
  • Growth medium containing serum
  • Cells
  • PBS ( appendix 2A)
  • BSA
  • Methyl‐β‐cyclodextrin (MBCD; Sigma)
  • recipeBSS (see recipe), optional
  • 10% Triton X‐100 in PBS
  • 60‐mm tissue culture dishes
  • Scintillation counter

Support Protocol 8: Cholesterol Repletion of Cells Using MBCD‐Cholesterol Complexes

  Materials
  • Cholesterol
  • Chloroform
  • Methanol
  • Methyl‐β‐cyclodextrin (MBCD; Sigma)
  • Growth medium without serum
  • 10‐ to 15‐ml glass tubes with Teflon‐lined screw caps
  • Nitrogen gas evaporator
  • 0.45‐µm syringe filters
  • 10‐ml syringes
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Figures

Videos

Literature Cited

Literature Cited
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