Chapter 3. Exploring Proteins and Proteo Flashcards
a technique for separating proteins. A mixture of proteins is electrophoresed in a pH gradient; each protein will migrate in the electrical field until it reaches its isoelectric point.
Isoelectric focusing
a separation technique based on size differences. A sample is applied to a column consisting of porous beads. Large molecules move through the column faster because they cannot enter the beads and, thus, have a shorter path to travel.
Gel-filtration chromatography
Ion-exchange chromatography in which a protein mixture is passed through a column containing a matrix bearing negative charges. Proteins bearing positive charges will bind to the column while those with negative charges will pass through the column.
Cation exchange
An assay for quantifying the presence of an antigen by using an enzyme linked to an antibody to the antigen.
Enzyme-linked immunosorbent assay (ELISA)
The process of removing small molecules from a solution containing a mixture of large molecules and small molecules. The mixture is placed in a bag made of a semipermeable membrane, which is then placed in a different solution. The membrane allows escape by the small molecules but not the large molecules.
Dialysis
Antibodies that are the products of many different populations of antibodyproducing cells.
Polyclonal antibody
a foreign substance that elicits the synthesis of an antibody.
Antigen
The mixture that results when the cell plasma membranes are disrupted prior to protein purification. The homogenate consists of fragments plasma membranes, organelles and the surrounding aqueous solution of protein, called the cytosol.
Homogenate
A technique for determining a proteins mass. The protein is evaporated to dryness in the presence of a volatile, aromatic compound that can absorb light at specific wavelengths. A laser pulse excites and vaporizes the matrix, converting some of the protein into the gas phase. Subsequent collisions enable the intermolecular transfer of charge, ionizing the protein. The newly formed ions then enter the mass analyzer, where they are distinguished on the basis of their mass-to-charge ratios.
Matrix-assisted laser desorption/ionization (MALDI)
Ion-exchange chromatography in which a protein mixture is passed through a column containing a matrix bearing positive charges. Proteins bearing negative charges will bind to the column while those with positive charges will pass through the column.
Anion exchange
(pI) the pH of a protein at which its net charge is equal to zero.
Isoelectric point
The chemical basis of Edman degradation. Phenyl isothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative that canliberated and identified, leaving an intact peptide shortened by one amino acid that can undergo another round of degradation.
Phenyl isothiocyanate
a protein purification technique that relies on the charge of proteins. Proteins are applied to an inert matrix to which is attached a charged moiety (e.g., a carboxylate group). Proteins will bind to the matrix with an affinity proportional to their content of the counterion (i.e., positive charges in the case of the carboxylate matrix).
Ion-exchange chromatography
a protein purification technique based on the high affinity many proteins have for specific chemical groups. Such groups are attached to an inert matrix, and the protein sample is applied; only those with an affinity for the groups will bind.
Affinity chromatography
An optical microscope capable viewing materials by reflection and absorption as well as visualizing fluorescent materials.
Fluorescence microscopy
A technique used to separate charged molecules, such as proteins and nucleic acids, which is based on the fact that such molecules will move at differing rates through a gelatinous material, such as polyacrylamide or agarose, when subjected to an electric field. Separation depends on factors such as net charge, size, and shape of the molecules.
Gel electrophoresis
