Ldh Lab Report

In this experiment, we had purified LDH isozyme from the clarified homogenate via the ammonium sulfate precipitation, affinity chromatography and size exclusion chromatography. Based on the result shown on the LDH table and on the SDS page, the most effective LDH purification step is the affinity chromatography. Even though the affinity purification step recovers the least amount of LDH (only 3% of LDH from the previous 65% cut), it shows the most purified enzyme without any contaminating proteins on the SDS PAGE. Since the effectiveness of purification is determined by both the recovery rate and the purity rate, other LDH intermediates do meet the qualifying condition. The 65% cut pellet has the highest recovery rate, 84% of the LDH of

However, the size exclusion chromatography experiment was not successful because no pellet was observed after the experiment. In the other words, the protein that we lost after the size exclusion trial was mostly LDH instead of contaminating proteins. The miscorrelation between the total protein and the total activity unit is the evidence to show that the LDH was lost during this purification step. If the contaminating proteins was being purified, these two values should be decreasing in the correlation to each other. Besides, the sudden drop of the specific activity from the affinity purified LDH to the size exclusion purified LDH also proves that we had washed more LDH than contaminated protein in this step. The decrease of fold purification in the size exclusion LDH intermediate again restated the problematic purification of LDH proteins in compared to the original clarified homogenate. Overall, the affinity chromatography is the most effective purification step to get rid of all the contaminating