6. Resume electrophoresis until the bromophenol blue in the serum albumi sample

Question

6. Resume electrophoresis until the bromophenol blue in the serum albumi sample has migrated to within 1 cm of the positive electrode end of the gel. Remove the gels from the unit and measure the distance of the proteins (in cm) from the sample wells. For long term storage of the gels, place them in destain solution for at least 1 hour and then perform step 6 on page 16. 7. (Optional) Suain and destain the agarose gels. 8. Measure the distance of the proteins (in cm) from the sample origin. Study Questions What is the molecular basis for the differences in the electrophoretic mobilities of the four proteins analyzed in this exercise? Relate your answer to the isoelectric points of these proteins that are given in Table 1-1. 1. 2. Explain how your results would have been affected if the electrophoretic separation was carried out at pH 10.5. 3. Explain how your results would have been affected if the separation was carried out at pH 6.0.

Explanation / Answer

Proteins are amino acid chains. These amino acids can have net positive negative and neutral charges (depending on the pH). More the length more the amount of these charges and hence they differ in their isoelectric points, the pH at which the protein has no net charge. Now in electrophoresis we generally neutralize the charges with SDS, the amount of SDS bound to the protein being proportional to the length of the protein and thus the charge now, being proportional to the length of the protein. Thus their mobility now becomes the function of their length.

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