Monday, February 16, 2009

Understanding of Protein Fingerprinting Technique

Breaking the protein molecules down into shorter fragments called peptides, Pauling subjected these fragments to another technique called protein fingerprinting by paper chromatography.

The technique of protein fingerprinting involves the following steps:

1) Extract and purify hemoglobin from sickle cell RBC and normal RBC separately in a clean test tube.

2) Digest these proteins with a commercial sample of trypsin separately under standard conditions. Trypsin is another type of serine protease that cleaves the peptide bond adjacent to a lysine or arginine residue in a protein molecule.

3) The cleaved peptides are subjected to paper electrophoresis under pH (pH 2.5) and dry the paper.

4) After electrophoresis they are subjected to paper chromatography perpendicular to the direction of electrophoresis using the solvent system water: butanol: acetic acid in the ratio 5:4:1. The peptides will separate depending on their partition coefficient, which further depends on their degree of hydrophobicity. The more hydrophobic peptide will move fast and the less hydrophobic will move slowly.

5) Remove the chromatographic paper and stain with ninhydrin.

6) Examine the peptide spots and compare with the standards.

When this procedure is applied to samples of normal and mutant (sickle) hemoglobin molecules (alpha and beta chains) that had been broken down into specific pep tides, all the spots are the same except for one crucial spot, which represents the difference between sickle cell and normal hemoglobin.

The protein fingerprinting or the peptide mapping developed for the molecular studies of sickle cell hemoglobin became a very powerful technique for the identification of protein samples from different sources. The peptide fingerprint of a protein from new sources can be compared with that of the standard protein and thus,. the variations can be identified or understood. This simple technique of peptide fingerprinting has given rise to another similar and more powerful technique: two-dimensional gel electrophoresis. This is a combination of two electrophoretic techniques - Isoelectric focusing and SDS-PAGE-in a series. First, the protein is subjected to isoelectric focusing, which is followed by 50S-PAGE in a direction perpendicular to the first. This technique was found to be very useful for proteomes studies, expression of protein profiles of cells grown under different conditions (for example, normal cells and diseased cells), and also easy identification of proteins in combination with mass spectrometry. All these technological advancements including amino acid sequencing have provided an enormous quantity of data, and that has given rise to computerized databases and homology searches and protein identification. All these have led to a generation of bioinformatics and computational biology.

Tags: Bio Technology, Bio Genetics, Proteins

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