What techniques are used to purify proteins?
● Protein purification strategies require the use of a highly sensitive and specific biochemical assay that identifies the target protein against a background of all other cellular proteins. Specific activity refers to the ratio of protein activity units to the total amount of protein in the sample.
● Differential centrifugation is used to fractionate proteins contained in a cell extract on the basis of the applied centrifugal force and length of centrifugation time. The resulting separation of pellet and supernatant can be used to isolate fractions enriched for proteins localized to nuclei, mitochondria, membranes, and cytosol.
● Column chromatography is a macromolecular separation technique that is essential to most protein purification strategies. Three common types of column chromatography are gel filtration chromatography, ion-exchange chromatography, and affinity chromatography.
● SDS-PAGE is a protein separation technique that uses sodium dodecyl sulfate (SDS) to give proteins a uniform charge-to-mass ratio, which allows their physical separation in polyacrylamide gel electrophoresis (PAGE) on the basis of molecular mass.
● Isoelectric focusing is a protein separation technique that is based on the differential isoelectric points of proteins. In the presence of a pH gradient and electric current, charged proteins migrate in the gel until they reach a point at which pI = pH and they carry no net charge.
What methods are used to sequence and synthesize oligopeptides?
● Edman degradation is a protein sequencing method that uses chemical labeling and cleavage, in conjunction with amino acid standards, to identify N-terminal amino acids sequentially. By fragmenting a polypeptide into small fragments using differential protease digestion, it is possible to deduce the amino acid sequence of the entire protein.
● Protein identification by mass spectrometry uses polypeptide fragmentation and high-resolution mass analysis to predict amino acid sequence on the basis of comparison of the measured mass to a predicted mass using computer algorithms and whole-genome databases.
● Solid-phase peptide synthesis is a method to generate oligopeptides of up to 25 amino acids using successive rounds of covalent linkage, washing, and deblocking to add amino acids one at a time to a resin-attached C-terminal amino acid. Once the oligopeptide is synthesized, it is released from the resin.
How are the three-dimensional structures of proteins determined?
● X-ray crystallography uses a focused X-ray beam directed at a protein crystal to obtain a diffraction pattern. From analysis of the position and intensities of the diffraction spots, an electron density map of the protein crystal can be calculated. Protein models are then built to match the regions of high electron density.
● NMR spectroscopy is used to determine the relative locations of atoms in a purified protein solution. From NMR spectra, a family of three-dimensional structures can be calculated that agrees with the data.
● Cryo-electron microscopy uses an electron beam to create projections of structures on a detector. Computational approaches are used to combine many different images to produce a model of the three-dimensional structure.
How are protein-specific antibodies made and what are their uses in biochemistry?
● Protein-specific antibodies are useful reagents in protein biochemistry because they identify target proteins with high affinity and specificity.
● Polyclonal antibodies are generated by injecting host animals with an antigenic protein or oligopeptide to induce an immune reaction and stimulate antibody production. Polyclonal antibodies are isolated from blood serum and represent a collection of antigen-selective antibodies that recognize multiple epitopes and are often affinity purified to remove nonspecific antibodies.
● Monoclonal antibodies are produced by injecting host animals with an antigenic protein or oligopeptide to induce an immune reaction and stimulate antibody production. Spleen cells are isolated from the immunized animal and fused with an immortalized tumor cell line to generate immortalized mouse hybridoma cells, which produce a single antibody that recognizes a particular epitope.
● Common applications of protein-specific antibodies include Western blotting, immunofluorescent staining of cells and tissues, enzyme-linked immunosorbent assays (ELISAs), and protein immunoprecipitation.