Reduced‐Scale Large‐Zone Analytical Gel‐Filtration Chromatography for Measurement of Protein Association Equilibria

Dorothy Beckett1

1 University of Maryland, College Park
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.5
DOI:  10.1002/0471140864.ps2005s18
Online Posting Date:  May, 2001
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Abstract

The proteasome plays a central role in eukaryotic cells since it is responsible for the degradation of specific proteins involved in a large range of cellular processes. Analysis of proteasome mechanisms of action, or in vitro reconstitution, or dissection of the complex biological pathways in which it partakes, requires a reliable source of pure active proteasome. Although the biologically relevant form of the proteasome is usually considered to be the 26S proteasome, this unit describes different methods for purification and study of both 26S and 20S proteasomes from Saccharomyces cerevisiaecells.

     
 
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Table of Contents

  • Basic Protocol 1: Reduced‐Scale Large‐Zone Analytical Gel‐Filtration Chromatography
  • Support Protocol 1: Data Analysis
  • Support Protocol 2: Determination of the Energetics and Stoichiometry of the Protein Assembly Process
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Reduced‐Scale Large‐Zone Analytical Gel‐Filtration Chromatography

  Materials
  • Gel‐filtration resin (e.g., Sephacryl S‐200, Amersham Pharmacia Biotech)
  • Column running buffer (e.g., 50 mM Tris⋅Cl, pH 7.5/200 mM NaCl, 20°C)
  • Column calibration standards (e.g., bovine serum albumin, hen egg ovalbumin, bovine pancreas ribonuclease A, bovine pancreas chymotrypsinogen A, glycine, and Blue Dextran 2000; Amersham Pharmacia Biotech)
  • Protein sample to be tested
  • Jacketed analytical glass columns (0.66‐cm i.d.; 10‐cm long; Omnifit) with end fitting and top column connection (Thomson Instrument; both with frits supplied by manufacturer)
  • 5/16‐ and 1/16‐in.‐i.d. Tygon tubing
  • Ring stands with clamps
  • Peristaltic pump
  • High‐performance liquid chromatography (HPLC) system with:
  •  HPLC pump (microbore), capable of delivering buffer at a flow rate of 0.152 ± 0.002 ml/min over long time periods (see Critical Paramameters)
  •  Teflon syringe‐loading sample injector (Rheodyne model 7125; Thomson Instrument)
  •  UV/visible detector and attached digital integrator of the detector signal
  • 1/16‐in.‐o.d. Teflon tubing
  • 0.25‐cm‐i.d. Teflon tubing
  • Column‐end‐fitting Teflon ferrules, drilled to accept 1/16‐in.‐o.d. Teflon tubing
  • Polymeric one‐piece nut and ferrules
  • Zero‐dead‐volume polymeric unions
  • 20‐µl Hamilton syringes
  • Refrigerated circulating water bath
  • Preweighed 1.5‐ml conical microcentrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
   Ackers, G.K. 1967. New calibration procedure for gel filtration columns. J. Biol. Chem. 242:3237‐3238.
   Ackers, G.K. 1975. Molecular sieve methods of analysis. In The Proteins, Vol. 1 (H. Neurath, R.L. Hill, and C. Roeder, eds.) pp. 1‐94. Academic Press, New York.
   Becker, G.W. 1988. Frontal boundary analysis in size exclusion chromatography of self‐associating proteins. In Aqueous Size Exclusion Chromatography (P.L. Dubin, ed.) pp. 375‐397. Elsevier Science Publishing, New York.
   Beckett, D., Koblan, K.S., and Ackers, G.K. 1991. Quantitative study of protein association at picomolar concentrations: The λ phage cI repressor. Anal. Biochem. 196:69‐75.
   Dubin, P.L. 1992. Problems in aqueous size exclusion chromatography. In Advances in Chromatography, Vol. 31 (J.C. Giddings, E. Grushka, and P.R. Brown, eds.) pp. 119‐151. Marcel Dekker, New York.
   Ip, S.H.C. and Ackers, G.K. 1977. Thermodynamic studies on subunit association constants for oxygenated and unliganded hemoglobins. J. Biol. Chem. 252:82‐87.
   Johnson, M.L. and Faunt, L.M. 1992. Parameter estimation by least squares methods. Methods Enzymol. 210:1‐36.
   Nenortas, E. and Beckett, D. 1994. Reduced‐scale large‐zone analytical gel filtration chromatography for measurement of protein association equilibria. Anal. Biochem. 222:366‐373.
   U.S. Government Printing Office. 1954. Tables of the Error Function and Its Derivative. N.B.S. Applied Mathematics Series 41, U.S. Government Printing Office. Washington, D.C.
   Valdes, R. and Ackers, G.K. 1979. Study of protein subunit association equilibria by elution gel chromatography. Methods Enzymol. 61:125‐142.
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