Separation / Purification
The first step in the study of a protein is to separate it from other cellular components, or to isolate it in a pure form. But how does a researcher separate different cellular components and different proteins? Techniques include centrifugation, chromatography and SDS-PAGE. Once the different cell components are separated out, proteins can be detected by their light absorption (protein absorbs 280 nm light, mainly by tryptophan and tyrosine), by immunological methods (ie, a Western blot or ELISA) or even gel electrophoresis.
Detection (Immunological Techniques)
How to detect a single protein from a mixture of proteins? ELISA, Immunoblot, Immunoprecipitation, etc.
Sequence and Structural Analysis
|Sequence||Edman degradation, mass spectrometry. Polypeptides are frequently fragmented prior to sequencing. The Edman degradation proceeds by identifying the amino-terminal residue and recycling the remaining peptide fragment through the Edman process. Mass spectrometry fragmentation occurs mainly at peptide bonds, and the charge is retained in one product.|
|Secondary structure||Circular Dichroism, FTIR. Circular dichroism (CD) measures amide (peptide bond) absorption of circularly polarized UV light. Ellipticity (∆ε) is the difference in absorption of left-handed and right-handed circularly polarized light. Different secondary structures show different patterns of ellipticity based on their secondary structure's composition of helix, sheet, turn and coil in their. Thereby, the protein’s CD spectrum can be ‘deconvoluted’ to estimate fractional contribution of helix, sheet, turn, and coil. Fourier transform infrared (FTIR) spectra show amide absorption of infrared light. Peak frequencies show bond stretching and bending, with one particular peak (referred to as Amide I Band) correlating the protein's secondary structure. It is then computed what approximate amount of helix, sheet and coil could have been lumped together to give rise to that particular shape of peak.|
|NMR, X-ray crystallography. Proteins have too many protons to be resolved by one-dimensional NMR, but two-dimensional NMR separates proton peaks and can reveal approximate distances between nearby atoms. NMR-derived distance constraints are used to calculate likely protein conformations, which are usually similar enough to provide a descriptive average estimated structure. While NMR measures atomic distance, X-ray crystallography reveals the layout of repeating electron density based on how protein crystals scatter x-rays. Computations reveal an electron-density map which can be used to position protein atoms, revealing the overall structure.|