Protein Synthesis Lab Report

In this section we will analyze which steps were the most effective ones in recovering LDH (percent yield) and in purifying LDH (fold purification). As we can see looking at the Total Protein column on Table 3, the most effective step with regard to the percent of remaining protein removed was affinity chromatography because it was able to remove 98.6% of the remaining proteins. In comparison to 81.93% removed during the 65% ammonium sulfate precipitation and 81.3% during the size exclusion. This means that the affinity chromatography removed a big percentage of contaminating proteins. However, removing this huge amount of protein left us with a small amount of LDH and cost us a considerable decrease on total activity. On the percent yield column, we can see that the purification step with the best recovery of LDH was the 65% cut, which gave us 11.04%, in comparison with just 2.26% during affinity chromatography. This shows
In addition, the sample could have been incubated incorrectly. This is a result that was not expected, since the addition of ammonium sulfate should have further purified the LDH. As a result, the most effective step in terms of LDH purity (fold purification) was the affinity chromatography with 1.67 fold in comparison to the 65% cut (0.611 fold). The worst step in regard to LDH purity was Size Exclusion (0 fold). However, those results would have been better if we did not have a problem with the column, as already mentioned above. We were able to visualize purity by analyzing the SDS-PAGE gel (Figure 8). We can clearly see in the gel, that the Clarified Homogenate is more pure than the 65% cut LDH. We should have been able to visualize that the best purification step was Affinity Chromatography, but during the experiment, instead of adding the 5x sample buffer, we added phosphate buffer.