Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Hence, they have different molecular structures, nutritional attributes and physicochemical properties. There are three major protein analysis techniques: protein separation, western blotting and protein identification.
1. PROTEIN SEPARATION
It is the process of separating or purifying proteins by placing them in a gel matrix and then observing protein mobility in the presence of an electrical field. It’s an important approach to studying protein function and the effect of a particular protein on development or a physical function by introducing it into an organism. The most commonly used technique for protein separation is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2. WESTERN BLOTTING
The western blot technique uses three elements to identify specific proteins from a complex mixture of proteins extracted from cells: separation by size, transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualize. The most common version of this method is immunoblotting. This technique is used to detect specific proteins in a given sample of tissue homogenate or extract. The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight. The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.
3. PROTEIN IDENTIFICATION
There are two methods that are commonly used to identify proteins: Edman Degradation and Mass Spectrometry.
Edman Degradation is a method of sequencing amino acids in a peptide. In this, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues. Protein Mass Spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles for determining masses of particles and the elemental composition of a sample of molecules as well as for elucidating the chemical structure of molecules such as peptides.
Protein complex analysis involves extensive interpretation of the structure and function of proteins, which are present in complex biological samples. Though recent protein complex analysis methods are efficient in identifying the structure of protein complexes, there are some limiting factors.
Phylogenetic analysis of proteins employs a combination of molecular and statistical approaches to infer or estimate relationships among individuals. It provides a credible method to explore the relationship between sequence similarity and function of proteins belonging to the same family.
Many proteins only contain a single domain, while others may have many domains. Some domains have a clearly defined function associated with them, like the Rossmann fold domain (also called coenzyme-binding domain). Such domains often “carry” their function with them when they get inserted into different proteins during evolution.
The physicochemical properties of a protein are determined by the analogous properties of the amino acids in it. Some amino acids are dextrorotatory, others are levorotatory. With the exception of a few small proteins (peptides) that occur in bacteria, the amino acids that occur in proteins are l-amino acids.
Protein–protein interactions (PPIs) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces, hydrogen bonding and the hydrophobic effect. Many are physical contacts with molecular associations between chains that occur in a cell or in a living organism in a specific biomolecular context.
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