Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients

Michael S. Marks1

1 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.3
DOI:  10.1002/0471143030.cb0503s00
Online Posting Date:  May, 2001
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Abstract

The molecular weight of a protein is a basic characteristic that can only be approximated by techniques such as gel filtration and electrophoresis. Zonal sedimentation analysis on sucrose gradients is a method for estimating the molecular mass of proteins and protein complexes under nondenaturing conditions. This unit includes protocols for preparing the appropriate gradients, for fractionation and separation of cell lysates on the gradients, for fractionation of the gradients themselves, and use of the results to calculate the molecular mass based on sedimentation coefficient and other parameters. There is also an additional protocol for differential sedimentation on gradients made with water and deuterium oxide to allow for direct determination of the partial specific volume of a protein or complexes.

     
 
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Table of Contents

  • Basic Protocol 1: Zonal Sedimentation Using Sucrose Gradient Formed by a Gradient Master
  • Alternate Protocol 1: Zonal Sedimentation Using Sucrose Gradients Formed by a Gradient Maker
  • Support Protocol 1: Use and Preparation of Common Molecular Size Makers
  • Basic Protocol 2: Fractionation by Puncture and Elution from the Bottom of the Gradient
  • Alternate Protocol 2: Fractionation by Peristalitic Elution from the Bottom of the Gradient
  • Alternate Protocol 3: Manual Fractionation by Removal of Material from the Top of the Gradient
  • Support Protocol 2: Determination of Sedimentation Coefficients by Extrapolation from Migration from of Standard Proteins
  • Support Protocol 3: Determination of the Value of ST,m
  • Support Protocol 4: Determination of the Value of Partial Specific Volume (υ)
  • Support Protocol 5: Determination of the Value of S20,w
  • Basic Protocol 3: Determination of Partial Specific Volume and Sedimentation Coefficient by Parallel Sedimentation in Media with Different Densities
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Zonal Sedimentation Using Sucrose Gradient Formed by a Gradient Master

  Materials
  • Buffer ingredients for stock solutions (e.g., HEPES, NaCl, Triton X‐100, protease inhibitors, sodium azide)
  • Sucrose, ultrapure
  • Sample in 100 to 250 µl volume (e.g., cell lysate)
  • Whatman no. 1 filter paper or 0.22‐µm filter
  • Refractometer
  • 14 × 89–mm centrifuge tubes for SW41 Ti rotor
  • Gradient Master (Biocomp), base unit, marker block, and holder for six 14 × 89–mm tubes
  • 10‐ml syringe fitted with a 6‐inch wide‐bore cannula
  • SW41 Ti rotor (Beckman) or equivalent
  • Ultracentrifuge (Beckman LS‐70M or equivalent)

Alternate Protocol 1: Zonal Sedimentation Using Sucrose Gradients Formed by a Gradient Maker

  • Gradient maker, small volume (e.g., 15 ml)
  • Microbore tubing
  • Glass capillary micropipet
  • Peristaltic pump or other pumping device (optional)

Support Protocol 1: Use and Preparation of Common Molecular Size Makers

  • Protein standards (e.g., Sigma)

Basic Protocol 2: Fractionation by Puncture and Elution from the Bottom of the Gradient

  Materials
  • Gradients to be eluted (see protocol 1 or protocol 2)
  • Sample tubes for fraction collector (e.g., 12 × 75–mm or microcentrifuge tubes)
  • Ring stand and clamp for tubes
  • 27‐G needle or commercial puncture device (e.g., Büchler)
  • Microbore tubing
  • Fraction collector (optional)

Alternate Protocol 2: Fractionation by Peristalitic Elution from the Bottom of the Gradient

  • Gradients to be eluted (see protocol 1 or protocol 2)
  • Mock gradient (same gradient as used for samples)
  • Peristaltic pump
  • Fraction collector with appropriate tubes (12 × 75–mm tubes or 1.5‐ml microcentrifuge tubes)
  • Glass microbore capillary pipet
  • Microbore tubing
  • Cap for centrifuge tubes with hole in the middle for microbore tubing
  • Rack for centrifuge tubes

Alternate Protocol 3: Manual Fractionation by Removal of Material from the Top of the Gradient

  • Manual pipettor and tips

Support Protocol 2: Determination of Sedimentation Coefficients by Extrapolation from Migration from of Standard Proteins

  Materials
  • Results from analysis of fractions for presence of sample of interest
  • Results from analysis of fractions for presence of standards
  • Calculator

Support Protocol 3: Determination of the Value of ST,m

  Materials
  • Results from analysis of fractions for presence of sample of interest
  • Results from analysis of fractions for presence of standard
  • Metric ruler
  • Calculator

Support Protocol 4: Determination of the Value of Partial Specific Volume (υ)

  Materials
  • Fractions eluted from sucrose gradients (see protocol 4 or protocol 5 or protocol 63)
  • Refractometer

Support Protocol 5: Determination of the Value of S20,w

  Materials
  • Deuterium oxide (heavy water, D 2O)
  • Additional reagents and equipment for zonal sedimentation using sucrose gradients (see protocol 1 or protocol 2), fractionation of gradients by elution (see protocol 4 or protocol 5Alternate Protocols 2 or protocol 63), and detection of proteins within fractions (see Strategic Planning)
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Figures

Videos

Literature Cited

Literature Cited
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   Coombs, D.H. and Watts, N.R. 1985. Generating sucrose gradients in three minutes by tilted tube rotation. Anal. Biochem. 148:254‐259.
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   Edelstein, S.J. and Schachman, H.K. 1967. The simultaneous determination of partial specific volumes and molecular weights with microgram quantities. J. Biol. Chem. 242:306‐311.
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   Gibbons, R.A. 1972. Physico‐chemical methods for the determination of the purity, molecular size and shape of glycoproteins. In Glycoproteins: Their Composition, Structure, and Function (A. Gottschalk, ed.) pp. 31‐128. Elsevier/North Holland, Amsterdam.
   Hall, P.K. and Roberts, R.C. 1978. Physical and chemical properties of human plasma α2‐macroglobulin. Biochem. J. 171:27‐38.
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   Martin, R.G. and Ames, B.N. 1961. A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Biol. Chem. 236:1372‐1379.
   Meunier, J.C., Olsen, R.W., and Changeux, J.P. 1972. Studies on the cholinergic receptor protein from Electrophorus electricus. Effect of detergents on some hydrodynamic properties of the receptor protein in solution. FEBS Lett. 24:63‐68.
   Millero, F.J., Ward, G.K., and Chetirkin, P. 1976. Partial specific volume, expansibility, compressibility, and heat capacity of aqueous lysozyme solutions. J. Biol. Chem. 251:4001‐4004.
   Neugebauer, J. 1994. A Guide to the Properties and Uses of Detergents in Biology and Biochemistry. Calbiochem‐Novabiochem International, La Jolla, Calif.
   Reisler, E., Haik, Y., and Eisenberg, H. 1977. Bovine serum albumin and aqueous guanidine hydrochloride solutions. Preferential and absolute interactions and comparison with other systems. Biochemistry 16:197‐203.
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Key References
   Helenius and Simons, 1975. See above.
  This is an excellent review covering the properties of detergents and how they bind to integral membrane proteins. It should be used as a guide for interpreting sedimentation experiments of integral membrane proteins in detergent solutions.
   Martin and Ames, 1961. See above.
  This is the seminal paper describing the technique of zonal sedimentation in sucrose gradients and supplies most of the basic background for understanding and performing the technique.
   van Holde, 1975. See above.
  This is an excellent review article covering the theoretical aspects of all of the major methods for sedimentation analysis of macromolecules.
Internet Resource
   http://131.202.97.21
  The Biocomp home page describes the Biocomp Gradient Master and Piston Gradient Fractionator with trumpet tip in detail.
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