Proteomics: Separation of Proteins using SDS-PAGE 

 

Proteins are the most abundant kind of macromolecules in human body. Proteins are made of long chains of amino acids and are the primary building blocks of tissues, functional molecules such as enzymes, channels in cell membrane, muscles for locomotion and dynamic activities, etc. In this lab the protein profiles of the muscle tissue from two different fish species were obtained. Differentiation of the different proteins present in the muscle tissue and quantification of these components would help identify the main constituents of the muscle tissue. 

 

Muscle consists of repeating subunits called sarcomeres. Sarcomeres in turn are composed of the actin filaments, myosin and tropomyosin. The movement of myosin along the actin would generate muscle contraction and the extent of this muscle contraction is harnessed by repeating units of tropomyosin along the actin filaments.

 

For analysing the red and white fish muscles, the samples were prepared for SDS-PAGE and the protein profiles were attained in Gel-Doc. Using the band patterns of protein prestained standard, the logarithm of molecular weights against the distance travelled on polyacrylamide gel can be graphed. The graph will constitute a standard calibration curve for extrapolation of the molecular weights of each of the bands on the protein profiles of red and white fish muscle. The intensity and molecular weights corresponding to each of the bands reveal the main components of the muscle. The bands corresponding to myosin, tropomyosin and actin have higher intensity comparing to other bands. However, this is only an educated guess about the identity of components corresponding to each band. This is because the distance migration of proteins depend on their structure and if the protein's structure is not fully disrupted during its preparation for SDS-PAGE then there is a possibility of a molecule with different identity to migrate the same distance as actin, myosin or tropomyosin. The techniques such as mass spectrometry can reveal the true identity of each of the bands.