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Protein Fingerprinting Principle and Procedure

Protein fingerprinting is an analytical technique for protein identification. Each protein has a unique peptide map and hence swerves as fingerprint for the protein. It involves the chemical or enzymatic treatment of a protein resulting in the formation of peptide fragments followed by separation and identification of the fragments in a reproducible manner. It is a powerful test that is capable of identifying single amino acid changes resulting from events such as errors in the reading of cDNA sequences or point mutations.
Protein Fingerprinting Procedure
Four major steps are necessary for the development of the procedure:
2. Selective cleavage of the peptide bonds
protein fingerprinting
3. Chromatographic separation of the peptides
4. Analysis and identification of the peptides.
A test sample is digested and assayed in parallel with a reference standard or a reference material. Complete cleavage of peptide bonds is more likely to occur when enzymes such as endoproteases (trypsin) are used, instead of chemical cleavage reagents.
Case study: Would you expect hemoglobin having Val substituting Glu at position of Beta chain to cause sickle cell RBCs?
Protein Fingerprinting steps involved:
  • purified normal/abnormal protein like Hemoglobin (Hb) is taken in to two separate test tubes.
  • enzymatic (like trypsin) treatment is given to both proteins.
  • trypsin breaks the peptide bond of lysine and arginine.
trypsin cleavage
  • Fragments so obtained are subjected to electrophoresis.
  • All the peptides will be similar except the mutated one.
 biochemistry of sickle cell anemia
Substitution of valine for glutamic acid will alter the structure of Hemoglobin (Hb) and result in deformation of the cell causing sickle cell anemia.

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