Size‐Exclusion Chromatography with On‐Line Light Scattering

Tsutomu Arakawa1, Jie Wen2

1 Alliance Protein Laboratories, Inc., Thousand Oaks, 2 Amgen Inc., Thousand Oaks
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.6
DOI:  10.1002/0471140864.ps2006s25
Online Posting Date:  November, 2001
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Abstract

This unit describes the use of size‐exclusion chromatography with on‐line light scattering, UV absorbance, and refractive index detectors (SEC‐LS/UV/RI) to determine: (a) the molecular weight of simple proteins containing no carbohydrates, (b) the molecular weight of glycoproteins, and (c), most importantly, the molecular weight and stoichiometry of protein‐protein complexes or protein‐carbohydrate complexes. Multiangle light scattering is also discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Using Refractive Index and Light Scattering to Calculate the Molecular Weight and Degree of Self‐Association of Proteins Containing no Carbohydrates (Two‐detector Method)
  • Alternate Protocol 1: Combination of LS and UV Detectors to Calculate the Molecular Weight of Nonglycosylated Proteins (Two‐detector Method)
  • Alternate Protocol 2: SEC/LS for Large Proteins: Debye Analysis
  • Alternate Protocol 3: Absolute Molecular Weight Calibration Method
  • Basic Protocol 2: Calculating the Stoichiometry of a Protein‐Protein Complex Containing no Carbohydrates Using LS/RI
  • Alternate Protocol 4: Calculating the Stoichiometry of Protein‐Protein Interactions for Nonglycosylated Proteins Using LS/UV
  • Basic Protocol 3: Calculating the Molecular Weight of Glycoproteins or Protein Conjugates Using Three Detectors
  • Basic Protocol 4: Determining the Stoichiometry of a Protein‐Protein Complex Containing Carbohydrates
  • Support Protocol 1: Sample Analysis: Receptor‐Ligand Interactions
  • Basic Protocol 5: Analysis of Protein‐heparin Interactions Using Three Detectors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Using Refractive Index and Light Scattering to Calculate the Molecular Weight and Degree of Self‐Association of Proteins Containing no Carbohydrates (Two‐detector Method)

  Materials
  • Protein solutions of unknown molecular weight, at ∼1 mg/ml
  • recipeCalibration standards (also see recipe): ∼1 mg/ml ribonuclease, ovalbumin, and BSA in column buffer
  • recipeElution (column) buffer (see recipe and unit 8.3)
  • Size‐exclusion (gel‐filtration) chromatography column (unit 8.3)
  • HPLC apparatus (units 8.3 & 8.7) including light‐scattering (miniDawn, Wyatt Technology) and refractive index (Agilent 1047A) detectors
  • Additional reagents and equipment for gel‐filtration chromatography (unit 8.3) and HPLC (unit 8.7)
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Figures

Videos

Literature Cited

Literature Cited
   Arakawa, T. and Wen, J. 2000. Refractive index of proteins in aqueous NaCl. Anal. Biochem. 280:327‐329.
   Arakawa, T., Yphantis, D.A., Lary, J.W., Narhi, L.O., Lu, H.S., Prestrelski, S.J., Clogston, C.L., Zsebo, K.M., Mendiaz, E.A., Wypych, J., and Langley, K.E. 1991. Glycosylated and unglycosylated recombinant‐derived human stem cell factors are dimeric and have extensive regular secondary structure. J. Biol. Chem. 266:18942‐18948.
   Arakawa, T., Langley, K.E., Kameyama, K., and Takagi, T. 1992. Molecular weights of glycosylated and nonglycosylated forms of recombinant human stem cell factor determined by low‐angle laser light scattering. Anal. Biochem. 203:53‐57.
   Arakawa, T., Wen, J., and Philo, J.S. 1994. Stoichiometry of heparin binding to basic fibroblast growth factor. Arch. Biochem. Biophys. 308:267‐273.
   Arakawa, T., Holst, P., Narhi, L.O., Philo, J.S., Wen, J., Prestrelski, S.J., Zhu, X., Rees, D.C., and Fox, G.M. 1995. The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: Effects of acidic amino acid substitutions J. Prot. Chem. 14:263‐274.
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   Horan, T., Wen, J., Arakawa, T., Liu, N., Brankow, D., Hu, S., Ratzkin, B., and Philo, J.S. 1995. Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3. J. Biol. Chem. 270:24604‐24608.
   Horan, T., Wen, J., Narhi, L., Parker, V., Arakawa, T., and Philo, J. 1996. Dimerization of the extracellular domain of granuloycte‐colony stimulating factor receptor by ligand binding: A monovalent ligand induces 2:2 complexes. Biochemistry. 35:4886‐4896.
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   Philo, J., Talvenheimo, J., Wen, J., Rosenfeld, R., Welcher, A., and Arakawa, T. 1994. Interactions of neurotrophin‐3 (NT‐3), brain‐derived neurotrophic factor (BDNF), and the NT‐3.BDNF heterodimer with the extracellular domains of the TrkB and TrkC receptors. J. Biol. Chem. 269:27840‐27846.
   Philo, J.S., Aoki, K.H., Arakawa, T., Narhi, L.O., and Wen, J. 1996a. Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: One high‐affinity and one low‐affinity interaction. Biochemistry 35:1681‐1691.
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