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Biophysical Analysis and Characterization

The Center of Excellence for Biophysical Characterization and Analysis possesses world-class equipment and analytical methods for biophysical analysis and characterization for proteins, including higher-order structural characterization, monomer/aggregates species determination, free-sulfhydryl content determination, and thermal stability characterization. Below are additional details on the methods and their intended purpose.

  • APPLICATION: Characterization of protein secondary and tertiary structures.

 

  • INTRODUCTION: Circular dichroism (CD) measures the differential absorption of left- and right-handed circularly polarized light in an optically active substance. Far-UV CD (190-250 nm) can reveal the protein secondary structure characteristics. Near-UV CD (250-350 nm) provides information on the protein tertiary structures. By comparing the CD spectra of two protein samples, the similarity between the samples can be evaluated.

  • APPLICATION: Characterization of protein secondary structures.

 

  • INTRODUCTION: Fourier Transform Infrared (FTIR) spectroscopy can be used to obtain information about the vibrational states of molecules. The major IR bands for proteins and polypeptides include the amide I (C=O stretching) and amide II (C-N stretching) vibrations. Different secondary structures (α-helix and β-sheet) exhibit characteristic frequencies and intensities in the amide I band region due to the differences in the hydrogen bonds in these structures. FTIR method can provide the percentage of secondary structures more accurately and easily.

  • APPLICATION: Determination of protein monomer and aggregates molecular weight.

 

  • INTRODUCTION: Size exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS) detector can separate proteins based on size and then measure the molecular weight of the separated monomer and aggregates.. This method is able to detect molecular weight across awide size range and with high sensitivity.

  • APPLICATION: Determination of protein molecular weight and aggregation states in native solution.

 

  • INTRODUCTION: Analytical ultracentrifugation (AUC) is a technique for the characterization of protein homogeneity, determination of aggregates and protein-protein interactions. Sedimentation coefficient is the key parameter for the aggregation analysis and used in both sedimentation velocity (SV) and sedimentation equilibrium (SE) methods. Multiple detection methods may be used in this technology, including absorbance and interference.

  • APPLICATION: Determination of concentration of amino acids in cell culture medium or after protein/peptide hydrolysis.

 

  • INTRODUCTION: Waters AccQ-Tag Pre-column derivatization method is applied for amino acid concentration determination with high accuracy and sensitivity (pmol level).


  • APPLICATION: Determination of protein/polypeptide extinction coefficient (EC).

 

  • INTRODUCTION: The protein absorbance can be measured by ultraviolet spectrophotometry at 280 nm, and its concentration (C) can be determined by quantitative Amino Acid (AA) analysis. The extinction coefficient (ɛ) of a protein can be determined based on Beer-Lambert’s Law.


  • APPLICATION: Determination of free sulfhydryl content in protein samples.

 

  • INTRODUCTION: Disulfide bonds play a key role in protein structural stability. Mismatched disulfide bonds and improper folding of protein may occur due to  free SH groups in the proteins. Free sulfhydryl content may be measured using a commercial kit (Measure-iT ™ Thiol Assay Kit).


  • APPLICATION: Determination of particle sizes, PDI and Zeta-potential of proteins.

 

  • INTRODUCTION: Dynamic light scattering (DLS) is to measure the particle size by illuminating the particles with a laser and analyzing the intensity fluctuations in the scattered light. Zetasizer Nano was used in DLS analysis to provide information such as size, PDI (Polydispersity index) and/or Zeta potential .


  • APPLICATION: Characterization of protein thermal stability.

 

  • INTRODUCTION: DSC is a thermodynamic technique. Through the measurement of heat capacity of sample during temperature changing, the thermal stability of protein is evaluated.
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