PROTEIN ANALYSIS TECHNIQUES - IIT JAM IIT JAM Notes | EduRev

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Explain Zone electrophoresis method of amino acid analysis.?
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PROTEIN ANALYSIS TECHNIQUES

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 physiochemical properties. There are three major protein analysis techniques, protein separation, western blotting and protein identification.

1. PROTEIN SEPARATION

  • SDS-PAGE: This method separates proteins mainly on the basis of molecular weight as opposed to charge or folding. It is a technique that is widely used in biochemistry, forensics, genetics and molecular biology.

  • Isoelectric Focussing: In this method, different molecules are separated by their electric charge differences. This technique is a type of zone electrophoresis that is usually performed in a gel and takes advantage of the fact that a molecule’s charge changes with the pH of its surroundings.

    Chromatic Methods: There are two chromatic methods frequently used for protein separation – high-performance liquid chromatography and thin-layer chromatography. Both these methods are particularly useful adjuncts to gel-based approaches. Although chromatography is a common technique in biochemistry laboratories used for purification, identification and quantification of protein mixtures, laser diffraction is traditionally used for pre-column size and polydispersity management.

    Two-dimensional Gel Electrophoresis: This is a powerful gel-based method commonly used to analyse complex samples in the interest of characterising the full range of proteins in the sample, not just a few specific proteins.

2. WESTERN BLOTTING

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.

  • 1. Edman Degradation: Developed by Pehr Edman, this is a method of sequencing amino acids in a peptide. Here, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

  • 2. Mass Spectrometry: An analytical technique that measures the mass-to-charge ratio of charged particles, mass spectrometry is used for determining masses of particles and the elemental composition of a sample of molecule as well as for elucidating the chemical structure of molecules such as peptides.

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