Determination of Molecular Weight by Sds-Page
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Jordi Lanis
03-31-05
Determination of Molecular Weight by SDS-PAGE
Introduction-
In the SDS-PAGE method, the proteins are subjected to passage through a polyacrylamide gel with varying percentages of the polyacrylamide material that will determine the rate at which the sample will pass through the gel being hindered by friction. Before this is done, the sample will be mixed with some of the solution containing SDS (sodium dodecyl sulfate). This compound dissociates in solution yielding the negative large ions that will be useful for the purpose of the electrophoresis. Since the Alkaline Phosphatase still has some positively charged groups on it, even though it has a net negative charge in the loading buffer, the SDS negative ions will surround these positively charged groups and make the protein a large negative mass of a molecule. This will be the factor that will guide it through the gel under voltage that will be applied to the gel. There will also be a dye in the sample mixture that is much smaller than the protein samples in the sample mixture. This will give us a reference of when to stop the current and a reference for calculating to the relative mobility of each band that will appear in the gel after electrophoresis. For our experiment, we mixed 7 microL of the loading buffer with 21 microL of the protein sample in an eppendorf tube. This was done for the stages 1, 3, and 4 enzymes of the alkaline phosphatase purification experiment since the stage 2 enzyme was too dilute. After heating the tubes for 5 minutes at 95 degrees, they were ready to be loaded into the gel. In the meanwhile, the TA prepared the gel electrophoresis apparatus. The gel was a 10 % polyacrylamide gel, which means that the material would be traveling a bit faster than said in the text book. The running buffer was composed of glycine, and Tris-HCL with a pH of 8.8, while the pH of the gel is at 6.8. In each of the gels (there were 2), the protein ladder was loaded into the first well and the commercially purified alkaline phosphatase was loaded into the second well. Then the samples of the groups were loaded into the gel in the order of the increasing group number and the order of the increasing sample number. After running the gel in 200 V for about 20 minutes, the electrophoresis process was finished. The TA stained and destained the gels and gave the results. The last three samples on the second gel are ours.
Results and observations-
The results of the gel electrophoresis were not as clear as one would have expected. The resolution of the dye was rather low for the protein bands in the final stained gel. Some of the bands can be barely distinguished especially, the stage 4 enzyme sample. The relative mobility of the markers in the latter was calculated by measuring the distance they traveled and dividing them by the distance that the dye traveled. The relative mobility of the bands is as follows:
.288
.376
.464
.664
.816
.968