Isolation of Platelet Granules

Juliane Nießen1, Gabriele Jedlitschky1, Andreas Greinacher2, Heyo K. Kroemer1

1 Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst‐Moritz‐Arndt‐University, Greifswald, Germany, 2 Department of Immunology and Transfusion Medicine, Ernst‐Moritz‐Arndt‐University, Greifswald, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.35
DOI:  10.1002/0471143030.cb0335s46
Online Posting Date:  March, 2010
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Abstract

Functional analysis of platelet intracellular structures requires isolation and purification of these cellular compartments. With regard to the function of platelets, both, dense (delta) and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited. This unit describes two protocols for isolation and purification of platelet granule structures. The Basic Protocol describes a new technique based on immunolabeling with target‐specific antibodies followed by magnetic sorting, whereas the Alternate Protocol describes the more traditional procedure based on differential centrifugation and density‐based sedimentation. For both methods, the degree of granule purification can be most easily determined by immunoblotting using various antibodies that recognize structure‐specific proteins. The immunomagnetic sorting method is especially good for studies requiring highly purified material (e.g., for the identification of specific transporters and receptors). Curr. Protoc. Cell Biol. 46:3.35.1‐3.35.14. © 2010 by John Wiley & Sons, Inc.

Keywords: human platelets; subcellular fractionation; magnetic sorting; sucrose density gradient; granule‐structures; immunopurification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Platelet Granules by Immunomagnetic Sorting
  • Elute Target Protein from the Magnetic Bead‐Complex
  • Support Protocol 1: Elute Target Protein from the Magnetic Bead Complex by Denaturing Elution
  • Support Protocol 2: Elute Target Protein from the Magnetic Bead Complex by Mild Elution with Citric Acid
  • Alternate Protocol 1: Isolation of Platelet Granules using a Density Gradient
  • Support Protocol 3: Characterization of Platelet Subcellular Fractions by Immunoblot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Platelet Granules by Immunomagnetic Sorting

  Materials
  • Adenine‐citrate‐dextrose‐anticoagulant (ACD‐A; code RDB8652, Baxter Healthcare)
  • Platelet‐rich plasma (PRP)
  • Washing buffer A (see recipe)
  • Apyrase (Sigma)
  • Hirudin (Pharmion)
  • Tyrode buffer (see recipe)
  • Washing buffer B (see recipe)
  • Homogenization buffer (see recipe)
  • 1000× protease inhibitors (stock solution, see recipe) or Boehringer Complete protease inhibitor (Roche Applied Science)
  • Liquid nitrogen
  • Tris/sucrose buffer (see recipe)
  • Target‐specific primary antibody (see Table 3.35.1)
  • Dynabeads M‐280 Sheep anti‐rabbit IgG (Invitrogen)
  • Dynabeads Pan anti‐mouse IgG (Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Centrifuge
  • 10‐ml polystyrol centrifuge tubes
  • 37°C incubator
  • Refrigerated Hettich Rotixa 50 RS Centrifuge (Hettich) with 4256 swing‐out rotor (or equivalent)
  • Loose‐fitting Dounce homogenizer, 15‐ml capacity (Wheaton)
  • Beckman ultracentrifuge with 70.1Ti rotor and corresponding polycarbonate ultracentrifuge tubes (12‐ml or equivalent)
  • Tight‐fitting Dounce homogenizer, 15‐ml capacity (Wheaton)
  • Teflon pestle glass homogenizer (Glas‐Col)
  • DynaMag‐2 magnet (Invitrogen)
    Table 3.5.1   Materials   Antibodies Used for Immunomagnetic Isolation and Immunoblotting of Platelet Granules a   Antibodies Used for Immunomagnetic Isolation and Immunoblotting of Platelet Granules

    Performance with
    Antigen Name and isotype Reactivity Immunomagnetic isolation Immunoblotting Supplier
    MRP4 SNG rabbit Human (X) X NC
    M 4I‐10 rat Human NT X Axxora
    GPIb Gi27 mouse Human X (X) NC
    GPIIb/IIIa Gi5 mouse Human X (X) NC
    P‐selectin CD62P mouse Human X (X) Beckman Coulter
    LAMP2 Mouse Human (X) X Santa Cruz
    GPIb CD42Iβ goat Human NT X Santa Cruz
    vWF Goat Human NT X Santa Cruz
    PDI Mouse Human (X) X ABCAM
    P‐selectin Goat Human (X) X Santa Cruz

     aAbbreviations: X, best performance; (X), satisfactory performance; NT, not tested; NC, not for sale; PDI, protein disulfide isomerase.
NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature.

Support Protocol 1: Elute Target Protein from the Magnetic Bead Complex by Denaturing Elution

  Materials
  • Platelet fraction–Dynabead complex (see the protocol 1)
  • 4% (w/v) SDS ( appendix 2A)
  • 95°C heating apparatus
  • DynaMag‐2 magnet
  • −80°C freezer
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)

Support Protocol 2: Elute Target Protein from the Magnetic Bead Complex by Mild Elution with Citric Acid

  Materials
  • Target protein–Dynabead complex (see the protocol 1)
  • 0.1 M citric acid
  • 1 M NaOH
  • DynaMag‐2 magnet
  • −80°C freezer

Alternate Protocol 1: Isolation of Platelet Granules using a Density Gradient

  Materials
  • Platelet‐rich plasma (PRP)
  • Washing buffer B (see recipe)
  • Homogenization buffer (see recipe)
  • 1000× protease inhibitors (stock solution; see recipe) or Boehringer Complete protease inhibitor (Roche Applied Science)
  • Tris/sucrose buffer (see recipe)
  • 60% (w/v) sucrose solution in 5 mM EDTA, pH 7.4
  • Refrigerated Hettich Rotixa 50 RS centrifuge (Hettich) with 4256 swing out rotor (or equivalent)
  • Ultrasonic processor UW60 (Bandelin Electronic)
  • Loose‐fitting Dounce homogenizer, 15‐ml capacity (Wheaton)
  • 15‐ml centrifuge tubes
  • Ultracentrifuge (Beckmann) with 70.1Ti fixed‐angle rotor and a swing‐out rotor (Beckmann SW40 or Sorvall TH‐641) and corresponding polycarbonate ultracentrifuge tubes (or equivalent)
  • Tight‐fitting homogenizer Dounce, 15‐ml capacity (Wheaton)
  • Teflon pestle glass homogenizer (Glas‐Col)
NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature, except when noted otherwise.
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Figures

Videos

Literature Cited

Literature Cited
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