10. If a native protein agarose gel electrophoresis is run under conditions wher
ID: 213188 • Letter: 1
Question
10. If a native protein agarose gel electrophoresis is run under conditions whereby a protein is at its pl, what factors would affect the migration of this protein through the gel? 11. What if that protein was first heated at 95 degrees C for 5 minutes prior to loading it in the wells of the gel? What factor(s) would determine its migration? 12. Why did some of your samples migrate "up" the gel instead of toward the positive electrode Would you expect the samples that migrated "up" to have more or less aspartate amino acids- why?Explanation / Answer
Please find the answers below:
Answer 10: The migration of protein in SDS PAGE depends upon their molecular weight when the proteins are present in their native linear polypeptide structure. Under such conditions, if the protein remains at a pH which is equal to the pI, the movement will depend solely upon the molecular weight since the driving force over here would be only their mass. This is because the protein would bear a net neutral charge at its pI and hence no movement would be drive by the charge.
Answer 11: When a protein is heated, it gets degraded into its primary structure i.e. a linear polypeptide which does not bear any rotations and other secondary structures. Thus, this linear polypeptide would be easily resolved onto the gel based upon its molecular weight. However, the charge of the protein would also play role in the movement since all amino acids bear a different charge. Collectively, two factors i.e. molecular weight and total charge would determine the movement of protein onto the gel.
Answer 12: As discussed above, the molecular weight as well as the charge determine the resolution of the protein on a gel. Generally, proteins move from negative to positive electrode on a gel but depending upon charge, the movement can be proceeded in opposite direction as well. This happens when the protein itself contains large amount of negatively charged amino acids suchas aspartic acid.
Thus, if a protein moves towards positive electrode, it is expected to contain very high amount of aspartate. This is because aspartate itself is a highly negatively charged amino acid and it would promote the protein to move towards positive electrode.
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