Protein And Mutant Alleles Analysis

Analysis of Proteins and Mutant Alleles Assignment
1. Determination of molecular weights of unknown proteins:
a. Prepare a HAND-DRAWN semi-log graph of your standards from the SDS- PAGE gel. (see attached graph).

b. Use the standard curve prepared in part (a) to determine the molecular weights of all protein bands in each of your three unknown samples (B, C, and D).
• Molecular weights of protein bands in each of the three unknown samples B, C, and D were determined using the standard curve. On the Y-axis, the corresponding migration distance (cm) was found and traced to meet the line of the standard curve. This point was then traced to meet the corresponding place on the x-axis, in which molecular weight of known samples were plotted.

A mutation in normal hemoglobin involving substitution of the amino acid valine for glutamic acid on the beta chains is characteristic of hemoglobin present in patients who suffer with sickle cell anemia. The point mutation in sickle cell hemoglobin, glutamic acid and valine, disturbs the charge that is required to maintain the structure of hemoglobin molecules- in changing the primary sequence of amino acids, the net charge of the protein is changed, as well as the conformation. These changes can be observed in sickle cell hemoglobin, as detected using agarose gel electrophoresis and the resulting migration patterns. At basic pH (9.2), normal hemoglobin molecules hold a net negative charge, causing them to migrate to the anode in an agarose gel undergoing electrophoresis. At the same basic pH, Sickle cell hemoglobin molecule hold two less negative charges than normal hemoglobin molecules (because the charge is changed in sickle cell hemoglobin), thus, its’ electrophoretic behavior is changed- the more negatively charged sickle cell hemoglobin molecule will migrate a farther distance to the anode than will a normal hemoglobin

On agarose gel electrophoresis, the three hemoglobin variants associated with sickle cell disease have different migration patterns based on their charge and weight. Since it holds a more negative charge, a normal hemoglobin protein (SS) will migrate to the anode of an agarose gel electrophoresis and appear as a single band. The form of hemoglobin in sickle cell anemia (ss) will migrate to the anode as a single band as well, but at a slower rate (farther away from the anode in comparison to SS). Since a hemoglobin molecule with the sickle cell trait (Ss) is comprised of both normal hemoglobin and sickle cell hemoglobin, this hemoglobin will migrate to the anode as two separate bands (one band next to ss, and one band next to