Selective Precipitation of Proteins

Daumantas Matulis1

1 Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 4.5
DOI:  10.1002/0471140864.ps0405s83
Online Posting Date:  February, 2016
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Abstract

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described. © 2016 by John Wiley & Sons, Inc.

Keywords: protein precipitation; protein stability; protein purification; salting out; matrix ligand; coprecipitation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Selective Precipitation by Salting Out
  • Alternate Protocol 1: Selective Precipitation by Stepwise Salting Out
  • Basic Protocol 2: Selective Precipitation by Isoionic Precipitation: Column Method
  • Alternate Protocol 2: Selective Precipitation by Isoionic Precipitation: Dialysis Method
  • Basic Protocol 3: Selective Precipitation Using a Two‐Carbon (C2) Organic Cosolvent
  • Basic Protocol 4: Selective Precipitation Using C4 and C5 Organic Cosolvents
  • Basic Protocol 5: Selective Precipitation Using Protein Exclusion and Crowding Agents and Osmolytes
  • Basic Protocol 6: Selective Precipitation Using Synthetic and Semisynthetic Polyelectrolytes
  • Basic Protocol 7: Selective Precipitation Using Metallic and Polyphenolic Heteropolyanions
  • Basic Protocol 8: Selective Precipitation Using Hydrophobic Ion Pairing (HIP) Entanglement Ligands
  • Basic Protocol 9: Selective Precipitation by Matrix‐Stacking Ligand Coprecipitation
  • Basic Protocol 10: Selective Precipitation Using Di‐ and Trivalent Metal Cation Precipitants
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Selective Precipitation by Salting Out

  Materials
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Appropriate pH buffer
  • Ammonium sulfate
  • Additional reagents and equipment for dialysis (Phillips and Signs, ; Zumstein, )

Alternate Protocol 1: Selective Precipitation by Stepwise Salting Out

Basic Protocol 2: Selective Precipitation by Isoionic Precipitation: Column Method

  Materials
  • Cation exchanger: Amberlite IR‐120 H+ resin (Sigma or Aldrich)
  • Anion exchanger: Amberlite IRA‐400Cl resin (Sigma or Aldrich)
  • ∼0.5 and 1 M NaOH or KOH
  • 0.5 to 1 M HCl
  • 0.2 M acetic acid
  • 0.2 M ammonium hydroxide
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Plastic beakers
  • Large Büchner funnel
  • Whatman no. 1 filter paper
  • Chromatography column of appropriate size

Alternate Protocol 2: Selective Precipitation by Isoionic Precipitation: Dialysis Method

Basic Protocol 3: Selective Precipitation Using a Two‐Carbon (C2) Organic Cosolvent

  Materials
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Buffering system
  • Ethanol
  • Acetone
  • Centrifuge

Basic Protocol 4: Selective Precipitation Using C4 and C5 Organic Cosolvents

  Materials
  • Ammonium sulfate
  • Buffering system
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • C 4 organic cosolvent (e.g., t‐butanol, analytical grade)

Basic Protocol 5: Selective Precipitation Using Protein Exclusion and Crowding Agents and Osmolytes

  Materials
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Appropriate pH buffer
  • Precipitating agent: neutral polymer or osmolyte

Basic Protocol 6: Selective Precipitation Using Synthetic and Semisynthetic Polyelectrolytes

  Materials
  • Water‐soluble polyelectrolytes (Aldrich or Sigma)
  • Crude protein solution of interest
  • Test tubes
  • Glass or plastic cuvettes
  • Spectrophotometer

Basic Protocol 7: Selective Precipitation Using Metallic and Polyphenolic Heteropolyanions

Basic Protocol 8: Selective Precipitation Using Hydrophobic Ion Pairing (HIP) Entanglement Ligands

Basic Protocol 9: Selective Precipitation by Matrix‐Stacking Ligand Coprecipitation

  Materials
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Potential ligands (Fig.  ; also see Aldrich catalog)
  • Appropriate buffers
  • Dowex‐1Cl resin
  • Small test tubes or 2‐ to 3‐ml plastic microcentrifuge tubes
  • Vortex mixer
  • Centrifuge

Basic Protocol 10: Selective Precipitation Using Di‐ and Trivalent Metal Cation Precipitants

  Materials
  • Analytical Reagent (AR)‐grade metal salts (generally as chloride or nitrate)
  • Crude protein solution of interest, particle‐free (see discussion of Clarification in Strategic Planning)
  • Appropriate buffer(s)
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Figures

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Literature Cited

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