Purification and Analysis of Thrombospondin‐1

Karen O. Yee1, Mark Duquette1, Anna Ludlow1, Jack Lawler1

1 Beth Israel Deaconess Medical Center, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.10
DOI:  10.1002/0471143030.cb1010s17
Online Posting Date:  February, 2003
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Thromboapondin 1 (TSP‐1) is a trimeric matricellular protein that is expressed by many cells. It contains several different domains that allow it to participate in cell adhesion, cell migration, and cell signaling. Recently TSP‐1 has been shown to activate transforming growth factor β (TGF‐β) and to inhibit both angiogenesis and tumor growth. This unit contains protocols for the purification of TSP‐1 from platelet‐rich plasma and the purification of TSP‐1 proteolytic fragments.

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Table of Contents

  • Basic Protocol 1: Isolation of Thrombospondin‐1 from Human Platelets
  • Basic Protocol 2: Isolation of Proteolytic Fragments of TSP‐1
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Thrombospondin‐1 from Human Platelets

  • Platelet‐rich plasma
  • Baenziger A buffer (see recipe)
  • Baenziger B buffer (see recipe)
  • 1 M CaCl 2 ( appendix 2A)
  • 1 N NaOH (optional)
  • Thrombin
  • Diisopropyl fluorophosphate (DFP)
  • Heparin‐Sepharose CL‐6B (Amersham Pharmacia Biotech)
  • 0.15, 0.25, 0.55, and 2.0 M heparin‐Sepharose column buffers (see recipe)
  • Anti‐vitronectin immunoaffinity column: prepare in advance according to manufacturer's instructions using an Affi‐Gel Hz Immunoaffinity kit (Bio‐Rad) and anti–human vitronectin antibody (e.g., GIBCO/BRL)
  • Ammonium sulfate
  • 10% and 20% (w/v) sucrose gradient solutions (see recipe)
  • 15‐ and 50‐ml centrifuge tubes (conical bottom preferred)
  • Preparative centrifuge (Sorvall RC‐B3 or equivalent) and rotor (H4000 or equivalent)
  • 40‐ml Oak Ridge centrifuge tubes
  • High‐speed centrifuge (Beckman J2‐MC or equivalent) and rotor (JA‐20 or equivalent)
  • 1 × 12–cm chromatography column
  • Fraction collector and appropriate tubes
  • Spectrophotometer set at 280 nm
  • Gradient maker
  • 14‐ml ultracentrifuge tubes
  • Ultracentrifuge (Beckman LM‐80 or equivalent) and rotor (SW 41Ti or equivalent)
NOTE: Platelets are temperature sensitive and activated by untreated glass surfaces; therefore, they should be handled at room temperature in plasticware, and centrifuges and buffers should be warmed to room temperature before use.

Basic Protocol 2: Isolation of Proteolytic Fragments of TSP‐1

  • TSP‐1 (see protocol 1)
  • TBS (see recipe) containing 2 mM CaCl 2
  • 0.5 M EDTA ( appendix 2A)
  • Chymotrypsin
  • Diisopropyl fluorophosphate (DFP)
  • 0.8 × 3–cm column of immobilized soybean trypsin inhibitor (Pierce)
  • 0.8 × 3–cm column of heparin‐Sepharose CL‐6B (Amersham Pharmacia Biotech)
  • 0.15, 0.25, and 0.55 M heparin‐Sepharose column buffers (see recipe)
  • Centriplus centrifugal filter device (3000‐Da cutoff; Millipore)
  • 1.3 × 30–cm column of Sephadex G‐200
  • Fraction collector
  • Spectrophotometer set at 280 nm
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Literature Cited

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