A variety of chromatographic methods are performed. These may include size exclusion chromatography for native protein size, ion exchange, hydrophobic, reverse-phase based upon solubility separation, dye-binding and other chromatographic methods to characterize proteins.
Purity is assessed by SDS-PAGE and/or nondenaturing electrophoresis for molecular weight and disulfide polymerization. Isoelectric focusing can be performed for identifying the isoelectric point (pI) of a protein. To determine homogeneous pure proteins, N-terminal amino acid sequencing is used. Other methods can be developed upon request. Electrophoresis describes the migration of charged particles under the….
Epitope mapping is the identification of the specific regions of a protein recognized by the antibody binding site. The protein is clipped into fragments, or fragments are expressed recombinantly, and tested for binding by antibody. This narrows down the protein region of antibody binding. A series of synthetic peptides containing overlapping amino acid sequences based….
High Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques. It utilizes a liquid mobile phase to separate the components of a mixture by forcing the components (analytes) dissolved in solvent to flow through a chromatographic column under high pressure. The mixture is resolved into its components based on the degree….
Ion exchange chromatography separates proteins or peptides based on charge characteristics. The net surface charge of a protein or peptide determines its adsorption to oppositely charged groups immobilized on the ion-exchange medium. Proteins are multivalent anions or cations, and the charge of a protein depends on the pH of the environment. When the pH is….
Enzymes, antibodies and other proteins can be purified from natural sources such as cells, tissues or plants, or produced recombinantly in bacteria, yeast and mammalian cell culture or in the baculovirus insect cell system. Fermentation lot sizes vary up to 1gm of specific recombinant protein. The desired protein can be purified to homogeneity. Protein identity….
Multi-gram quantities of antibodies or proteins can be produced by suspension culture (125 mL to 15 Liter bioreactor flasks) in serum-free medium. Antibodies can be further purified by various methods.
The limit of solubility and adjustment of salt, pH and formulation to increase solubility are performed. Solubility in a variety of detergents are investigated to retain protein or enzyme activity and conformation when developing isolation methods from cell membranes.
There are a variety of strategies to stabilize proteins and enzymes. Storage of the proteins is investigated including lyophilization, flash freezing, normal freezing methods, and storage below 0oC with glycerol or DMSO like compounds. If kept at 2-8oC, the types of potential antibacterial reagents is determined. If kept at 2-8oC without antibacterial reagents, the method….