Protein Extraction and Structural and Functional Characterization

Protein Extraction

Protein Extraction 




Alkaline Extraction
  • Oakton pH 700 meter
  • ThermoScientific SnakeSkin dialysis tubing
  • Beckman Centrifuges

To extract protein from plants or alternative sources using alkaline methods

Ultrafiltration Assisted Salt Extraction
  • Sartorius Vivaflow 200 membrane ultrafiltration units
  • ThermoScientific SnakeSkin dialysis tubing
  • Beckman Centrifuges

To extract protein from plants or alternative sources by adding salt and using ultrafiltration

Enzyme Assisted Extraction
  • Mettler Toledo DL22 pH Stat Titrator Food and Beverage Analyzer

To enhance protein extraction methods with appropriate enzymes
Extract Structure function

 Structural Characterization




Protein Profile via One or Two-Dimensional Gel Electrophoresis
  • Densitometer to scan gels and determine protein band densities (BioRad)

To determine the protein profile under reducing and non-reducing conditions
To reveal polymerization and/or hydrolysis patterns, potential bonding mechanisms, and the prevalence of individual protein subunits

Molecular Aggregation via Surface Hydrophobicity
  • Bio-TEK Synergy HT

To determine protein unfolding and subsequent polymerization driven by hydrophobic interactions
To estimate hydrophobic surface area and changes throughout processing

Molecular Aggregation via Surface Charge (Zeta-Potential)
  • Malvern Zetasizer zeta potential and particle size analysis

To estimate the net charge density on the surface
To indicate any changes in surface hydrophilicity, protein unfolding, and possible interactions with macromolecules such as carbohydrates

Molecular Aggregation via Free Sulfhydryl Concentration
  • Chemical assay

To indicate any protein unfolding and to reveal free SH that were previously entrapped in the three dimensional structure of the protein
To determine the redox state of the protein

Particle size analysis of powders, liquids, and emulsions
  • LA-960 HORIBA laser scattering particle size distribution analyzer

To measure particle size of powders, liquids, and emulsions

Degree of Hydrolysis using OPA method
  • Bio-TEK Synergy HT

To determine the degree of hydrolysis and the extent of amine blocked by the Maillard reaction

Structural Denaturation using Differential Scanning Calorimetry (DSC)
  • Mettler Toledo DSC1 differential scanning calorimetry

To determine the onset temperature of denaturation and the degree of unfolding as affected by processing or modification
To estimate protein denaturation state

Protein secondary configuration by FTIR
To measure the distribution of secondary protein structures as affected by processing conditions or modification

Protein Tertiary Structures by Surface Enhanced Raman Spectroscopy (SERS)

To determine the distribution of tertiary protein structures and intramolecular interactions as affected by processing conditions or modification

Apparent Protein Aggregation State by Size Exclusion HPLC
  • Shimadzu LC-2050C 3D

To identify degree of polymerization, the extent of intermolecular aggregation with covalent and non-covalent linkages, and molecular weight of polymer distribution

Aggregation via Transmission-Scanning Electron Microscopy

To visualize protein and particle dynamics (aggregation morphology)

Chemical Deviation Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF)

To determine the individual protein subunit/peptide molecular weights, which can be used for identification of different protein subunits
To identify changes in proteins and peptides profile/distribution as affected by processing and/or enzyme modification
To monitor the site of modification

Chemical Deviation via LC/MS/MS

To separate and identify specific protein subunits

Structural Visualization via Confocal Laser Scanning Microscopy (CLSM)

To visualize protein-protein and protein-lipid interactions in dispersed systems while utilizing fluorescent probes
Functional Characterization

Functional Characterization




  • Oakton pH 700 meter
  • LECO FP 828

To determine the soluble protein in a protein-water dispersion by measuring the protein concentration of the dispersion before and after centrifugation to sediment insoluble proteins.

Oil-Binding Capacity
  • Chemical assay

To determine the amount of oil bound to the protein structure. Oil-binding capacity is related to a protein's surface hydrophobicity and influences its emulsification properties.

Water-Binding Capacity
  • Chemical assay

To determine the amount of water bound to protein via hydrogen or ionic bonds.

Gelation and Least Gelation Capacity
  • Texture Analyzer XT Plus

To determine a protein's ability to form a three-dimensional network with entrapped water. Gel strength measures the force required to rupture the gel. Least gelation concentration determines the lowest protein concentration at which a protein solution forms a heat-induced gel and holds its shape.

Foaming Capacity & Stability
  • Scilogex D500 and IKA RW 20 benchtop homogenizers

To determine a protein's ability in a protein solution to produce a foam after agitation. Foaming stability, on the other hand, refers to the ability of a protein foam to remain stable over time, and against gravitational or mechanical stresses.

Emulsification Capacity
  • Scilogex D500 and IKA RW 20 benchtop homogenizers

To determine the quantity of oil a certain amount of protein can emulsify. Oil is titrated into a protein solution and emulsion failure is measured by a decrease in viscosity.

Emulsification Activity Index and Stability
  • Beckman Coulter DU 800 UV/Visible scanning spectrophotometer with temperature regulated cells and enzyme mechanism software

To determine the amount of oil that can be emulsified per unit protein. Emulsification stability is given in the number of minutes that the emulsion will be stable.

Color Analysis
  • Minolta Chroma Meter

To determine L*a* b* values of protein ingredients as affected by extraction conditions, glycation, processing conditions, etc.