Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

  title={Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.},
  author={Brent S. Kendrick and Bruce A. Kerwin and B S Chang and John S. Philo},
  journal={Analytical biochemistry},
  volume={299 2},
Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein… Expand
Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors.
Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determinationExpand
Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection.
The studies reveal that different monoclonal immunoglobulin Gs and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis, and suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregation. Expand
Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering.
  • Hongping Ye
  • Chemistry, Medicine
  • Analytical biochemistry
  • 2006
The feasibility of size exclusion chromotography (SEC)-multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, andExpand
Analysis of Oligomeric and Glycosylated Proteins by Size-Exclusion Chromatography Coupled with Multiangle Light Scattering.
This chapter provides several examples of analysis of glycosylated protein conjugates to showcase the power of SEC-MALS, a union of two techniques that can help determine the absolute molecular stoichiometry, homo- and heteroassociation of sample components, the nature of protein conjugs, and the molar mass of single molecules and multisubunit complexes. Expand
Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance, and Refractive Index Detectors.
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Light scattering coupled with reversed phase chromatography to study protein self-association under separating conditions.
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Measurement of uncertainty in peptide molecular weight determination using size-exclusion chromatography with multi-angle laser light-scattering detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Abstract The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionizationExpand
Characterization of a protein conjugate using an asymmetrical-flow field-flow fractionation and a size-exclusion chromatography with multi-detection system.
Detailed characterization of a protein-PEG conjugate using two separation techniques, that is, asymmetrical-flow field-flow fractionation (AF4) and size-exclusion chromatography (SEC) and Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a complementary characterization technique. Expand
HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes
The primary intention of this unit is to describe how to apply the combination of high‐performance liquid chromatography SEC and SLS‐UV‐RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein‐detergent complexes. Expand


Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.
Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simpleExpand
Molecular weight determination and compositional analysis of dextran-protein conjugates using low-angle laser light scattering technique combined with high-performance gel chromatography.
The observations suggest that the increase in the hydrophobicity of the protein moiety as a result of partial denaturation and the introduction of the Hydrophilic dextran chain affords the conjugate an amphiphilic property. Expand
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This chapter presents the basic principle of measurement of the molecular weight of a solubilized membrane protein and describes the practical techniques involved in the measurement. Expand
Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant.
It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. Expand
On-line characterization of polyethylene glycol-modified proteins
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Subunit molecular weights of the (Na+,K+)-ATPase of canine renal outer medulla were estimated by the measuring system consisted of components connected in the following sequence: a TSK-gel G3000 SW column, a UV spectrophotometer, a low-angle laser light scattering photometer, and a differential refractometer. Expand
Molecular weight determination of phospholamban oligomer in the presence of sodium dodecyl sulfate: application of low-angle laser light scattering photometry.
The number of polypeptides constituting the oligomeric structure of canine phospholamban (a putative regulator of Ca(2+)-ATPase of cardiac sarcoplasmic reticulum) stable even in the presence ofExpand
Molecular weights of CTLA-4 and CD80 by sedimentation equilibrium ultracentrifugation.
This work combines several approaches to characterize the molecular weights of the extracellular domains of the glycoproteins CTLA-4 and CD80 using carbohydrate analysis, electrospray mass spectrometry, size exclusion chromatography, and analytical ultracentrifugation. Expand