Dye Affinity Chromatography

Earle Stellwagen1

1 University of Iowa, Iowa City
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.2
DOI:  10.1002/0471140864.ps0902s00
Online Posting Date:  May, 2001
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Abstract

Dye affinity chromatography is a protein purification procedure based on the high affinity of immobilized dyes for the binding sites on many proteins. It is a rapid, inexpensive, and versatile method that is applicable to the purification of crude cellular extracts. This unit presents protocol for the three types of dye affinity chromatography: negative chromatography, positive chromatography, and tandem chromatography. An initial protocol describes a chromatographic procedure in which a small volume of the protein mixture to be purified is applied to a series of miniature columns, each containing a different immobilized dye. Analysis of the flowthrough and bound material allows determination of the optimum dye material for largerā€scale purification. An alternate procedure describes a similar initial selection procedure using centrifugation instead of chromatography. A support protocol describes a simple procedure for immobilization of free dyes.

     
 
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Table of Contents

  • Basic Protocol 1: Selection of Components Using Chromatography
  • Alternate Protocol 1: Selection of Components Using Centrifugation
  • Basic Protocol 2: Negative Chromatography
  • Basic Protocol 3: Positive Chromatography Using Batch Elution
  • Alternate Protocol 2: Positive Chromatography Using Gradient Elution
  • Basic Protocol 4: Tandem Chromatography
  • Support Protocol 1: Immobilization of Reactive Dyes
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Selection of Components Using Chromatography

  Materials
  • Protein mixture to be purified
  • Appropriate application solvent (see step )
  • Series of immobilized dyes (Table 9.2.1 and protocol 7)
  • Concentrated solution of an appropriate elution reagent (see step )
  • Plastic disposable filtration unit with pore size ≤0.45 µm
  • Small plastic disposable chromatography tubes with bed volume at least 1 ml
    Table 9.2.1   Materials   Some Commercially Available Immobilized Dyes a   Some Commercially Available Immobilized Dyes

    Dye Immobilized dye Supplier b
    Reactive blue 2 Affi‐Gel blue gel Bio‐Rad
    Blue Sepharose CL‐6B Pharmacia Biotech
    Cibacron blue 3G‐A‐agarose ICN Biomedicals, Sigma
    Cibacron blue F3G‐A Pierce
    DyeMatrex blue A gel Amicon
    Reactive blue 4 Reactive blue 4‐agarose Sigma
    Reactive blue 72 Reactive blue 72‐agarose Sigma
    Reactive brown 10 Reactive brown 10‐agarose Sigma
    Reactive green 5 Reactive green 5‐agarose ICN Biomedicals, Sigma
    Reactive green 19 DyeMatrex green A gel Amicon
    Reactive green 19‐agarose Sigma
    Reactive red 120 DyeMatrex red A gel Amicon
    Reactive red 120‐agarose ICN Biomedicals, Sigma
    Red Sepharose CL‐6B Pharmacia Biotech
    Reactive yellow 2 Reactive yellow 2‐agarose ICN Biomedicals, Sigma
    Reactive yellow 3‐agarose Sigma
    Reactive yellow 3 DyeMatrex orange A gel Amicon
    Reactive yellow 13 Reactive yellow 13‐agarose Sigma
    Reactive yellow 86 Reactive yellow 86‐agarose Sigma

     aScreening kits containing between 5 and 40 immobilized dyes in miniature chromatography columns can be purchased from Affinity Chromatography, Amicon, Wako Pure Chemical, and Sigma. Alternatively, free reactive dyes such as those listed in Table 97.80.4711 can be purchased, immobilized, and prepared for chromatography as described in the protocol 7.
     bSupplier names and addresses are provided in suppliers appendix.

Alternate Protocol 1: Selection of Components Using Centrifugation

  Materials
  • Protein mixture to be purified
  • Application solvent, with and without 2 M NaCl
  • Immobilized dye selected for negative chromatography (see protocol 1 or protocol 2)
  • 0.02% (w/v) sodium azide
  • Empty chromatographic column with height/diameter ratio ≤5 (sintered glass funnel can suffice)

Basic Protocol 2: Negative Chromatography

  Materials
  • Protein mixture to be purified
  • Appropriate application solvent
  • Immobilized dye and elution solvent selected for positive chromatography (see protocol 1 or protocol 2)
  • Empty chromatographic column with height/diameter ratio >5
  • Fraction collector

Basic Protocol 3: Positive Chromatography Using Batch Elution

  • Empty chromatographic column having a height/diameter ratio of >5
  • A coated magnet and magnetic stirrer
  • Linear gradient maker (commercial or constructed from two beakers of the same size connected by a siphon)

Alternate Protocol 2: Positive Chromatography Using Gradient Elution

  Materials
  • Protein mixture to be purified
  • Appropriate application solvent
  • Immobilized dyes selected for negative and for positive chromatography ( protocol 1 or protocol 2)
  • Selected elution solvent
  • 2 M NaCl
  • 0.02% (w/v) sodium azide
  • Empty chromatographic column with height/diameter ratio >5 (two columns are required if positive chromatography is to be performed using batch elution)
  • Empty chromatographic column with height/diameter ratio ≥5 (if positive chromatography is to be performed using gradient elution)
  • Linear gradient maker (for gradient elution)
  • Fraction collector
  • Pipettors and disposable tips

Basic Protocol 4: Tandem Chromatography

  Materials
  • Reactive dye (Table 9.2.2)
  • Concentrated solution of KCl
  • Reactive chromatographic matrix: e.g., Sepharose CL‐4B or CL‐6B (Pharmacia Biotech) or other cross‐linked agarose
  • 4 M and 1 M NaCl
  • 10 M NaOH
  • 2 M NH 4Cl
  • Large sintered glass funnel
  • Plastic disposable filter with pore size of 0.45 µm
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Figures

Videos

Literature Cited

Literature Cited
   Clonis, Y.D., Stead, C.V., and Lowe, C.R. 1987. Novel cationic triazine dyes in protein purification. Biotechnol. Bioeng. 30:621‐627.
   Hogg, P.J. and Winzor, D.J. 1985. Effects of solute multivalency in quantitative affinity chromatography: Evidence for cooperative binding of horse liver alcohol dehydrogenase to blue Sepharose. Arch. Biochem. Biophys. 240:70‐76.
   Robinson, J.B. Jr., Strottmann, J.M., Wick, D.G., and Stellwagen, E. 1980. Affinity chromatography in nonionic detergent solutions. Proc. Natl. Acad. Sci. U.S.A. 77:5847‐5851.
   Robinson, J.B. Jr., Strottmann, J.M., and Stellwagen, E. 1981. Prediction of neutral salt elution profiles for affinity chromatography, Proc. Natl. Acad. Sci. U.S.A. 78:2287‐2291.
   The Society of Dyers and Colourists and the American Association of Textile Chemists and Colorists. 1971. Colour Index, 3rd edition. The Society of Dyers and Colourists, Bradford, England, and the American Association of Textile Chemists and Colorists, Research Triangle Park, NC.
Key References
   Clonis, Y.D., Atkinson, T., Bruton, C.J., and Lowe, C.R. 1987. Reactive Dyes in Protein and Enzyme Technology. Macmillan, New York.
  A comprehensive review of the preparation, immobilization, and utilization of reactive dyes for protein purification.
   Lowe, C.R., Burton, S.J., Burton, N., Stewart, D.J., Purvis, D.R., Pitfield, I., and Eapen, S. 1990. New developments in affinity chromatography. J. Mol. Recognit. 3:117‐122.
  A lively discussion of emerging developments.
   Scopes, R.K. 1994. Protein Purification, 3rd ed. Springer‐Verlag, New York.
  A useful discussion of immobilized dye affinity chromatography by the advocate of tandem chromatography.
   Stellwagen, E. 1990. Chromatography on immobilized reactive dyes. Methods Enzymol. 182:343‐357.
  An alternative discussion of the topic with more emphasis on strategy and less on detail.
   Stellwagen, E. 1993. Affinity chromatography with immobilized dyes. In Molecular Interactions in Bioseparations (T.T. Ngo, ed.) pp. 247‐255. Plenum Press, New York.
  A detailed review of the interaction of reactive blue 2 with a typical oligomeric protein, alcohol dehydrogenase.
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