SIZE-EXCLUSION CHROMATOGRAPHY WITH ONLINE LIGHT-SCATTERING, ABSORBENCY, AND REFRACTIVE-INDEX DETECTORS FOR STUDYING PROTEINS AND THEIR INTERACTIONS

Techniques of using size-exclusion chromatography (SEC) with on-line l ight-scattering, uv absorbance, and refractive index detectors to char acterize the polypeptide molecular weights of simple proteins or glyco proteins or to determine the stoichiometry of protein complexes are de scribed. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution po sition and can exclude the contributions from carbohydrates. When a pr otein or complex contains no carbohydrates, a two-detector method, i.e ., light scattering combined with refractive index, can be used to cal culate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of pol ypeptide alone. Finally, a self-consistent three-detector method is us ed to determine the stoichiometry of a protein complex containing carb ohydrates. Example applications for all these methodologies are descri bed. (C) 1996 Academic Press, Inc.