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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 cerevisiae cells .

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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

Figure 20.5.1 Schematic drawing of the setup used for pouring the reduced‐scale column for large‐zone analytical gel‐filtration chromatography. The lower column is to be packed and the upper is the reservoir for resin.

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Figure 20.5.2 Schematic representation of a small zone of protein run on the reduced‐scale gel‐filtration column. The peak shape should be nearly symmetrical as indicated in this figure.

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Figure 20.5.3 Characteristic large zone of oxyhemoglobin A_o , plateau [Hb dimer] = 5.62 µM in 100 mM Tris⋅Cl, pH 7.40 ± 0.01/100 mM NaCl/1 mM Na₂ EDTA at 21.5°C, run on Sephacryl S‐200. The centroid position, or V _c , is defined as the position on the elution volume scale of the solid vertical line that yields equal areas for regions A and B.

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Figure 20.5.4 Example of a calibration curve obtained from analyzing small zones of protein standards on a reduced‐scale analytical gel‐filtration column packed with Sephacryl S‐200 HR resin. The standards from largest to smallest are BSA, ovalbumin, chymotrypsin, and RNase A. The solid line represents the best fit of the data to Equation .

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Figure 20.5.5 Association curve for the oxyhemoglobin A_o (Hb) dimer‐tetramer equilibrium. Experimental fraction Hb dimer values (dots) were obtained at twelve micromolar Hb dimer concentrations. The solid line is simulated using the best resolved parameters determined by nonlinear least squares analysis.

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Literature Cited

Literature Cited
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