Purification Step Lab Report

For this type of experiment, we would generally see a decreasing trend for the total activity, total amount of protein, and percent yield and an increasing trend for specific activity and fold purification. These trends come about naturally when performing multiple purification steps. To determine the success of each purification step, the more important factors to look at are the total activity units, total protein amount, and percent yield. Looking at our purification table (table 7) our purifications were not very successful. While our total protein followed the normal decreasing trend, as it should have, our total activity unit, which affects our percent yield, was very off, in terms of a decreasing trend. Instead, the total activity
Errors that occurred during the affinity step that impacted the purity of our LDH was that we had to stop the column before we could get an absorbance rate of 0.1, which means that there was much of our purer LDH sample that we were unable to collect and use. Another thing to note is that our values for the affinity chromatography step were almost the same as the amount for the size exclusion chromatography step. The total activity was 96.50U and the percent yield was 29.76% for the size exclusion. The lack of purity and low recovery of our size exclusion chromatography could be due to the column beads not being packed and settled before we ran our sample through it, since the column leaked and the beads were rehydrated right before our lab. The total protein data shows that the size exclusion chromatography was not a very useful step in this process and did not remove many excess
This was shown through our SDS-PAGE gel (figure 7). The gel showed that our LDH sample matched that of standard #1. We were able to determine that this first standard was the heart muscle LDH isozyme, by comparing the pH of the gel (pH 9) to the pI of the isozyme, 5.5 (heart) and 8.7 (skeletal muscle). Since we know these charges, and the rule that when a pH is greater than pI it will move toward the positive and when a pH is less then the pI it will stay toward the negative, we can compare to see that pH 9 is greater than pI 5.5. This means that the heart muscle LDH isozyme will move towards the positive end, the bottom of the gel, thus it will move further from the well than the skeletal muscle LDH isozyme with it’s pI of 8.7, almost equal to that of the pH of the