Introduction
Phospholipase A. is an enzyme used by Leukocytes to destroy bacteria, by causing membrane permeability. In multiple purification steps, we must isolate the enzyme and prove our experiment is a success.
Results
Figure 1 shows Sephadex carboxymethyl ion exchange chromatography of a leukocyte’s cytoplasm. The 3 things being measured in this graph are: overall protein concentration, permeability-increasing activity(PI) and phospholipid A(PLA) activity. PLA activity is measured by the destruction of the phospholipids. Figure 2 shows the SDS-PAGE of the fraction number, which is the X-axis of Figure 1. In figure 2, a constant level of protein must be used through each lane of the SDS, in every step. The sample enzyme must be …show more content…
E has the highest peak, although it's mainly protein that is not of interest to us. There are 4 bands of protein and 2 band of considerable thickness. This means there are multiple proteins of the same size present. The peak F is considerably smaller, but there seems to be more purification. There are only 3 bands of protein and on is thick. PI is also coming into play at this peak, which means the membrane of the bacteria are being affected. Peak G has even more protein purification, with only 2 bands. The thick band is around 55KD which is where phospholipase A shows to move towards. At G, protein concentration is very low, while PI and PLA are evenly high. This shows where the most activity is and where our specific enzyme is located. Moving to H, we see little to no activity and overall protein concentration seems to be increasing. Table 1 shows the multiple step purification process of phospholipase A. Homogenate had a large protein concentration, which means that our sample is far from purification. The fold purity is only 1 and bacterial activity is high.
Phospholipase A. is an enzyme used by Leukocytes to destroy bacteria, by causing membrane permeability. In multiple purification steps, we must isolate the enzyme and prove our experiment is a success.
Results
Figure 1 shows Sephadex carboxymethyl ion exchange chromatography of a leukocyte’s cytoplasm. The 3 things being measured in this graph are: overall protein concentration, permeability-increasing activity(PI) and phospholipid A(PLA) activity. PLA activity is measured by the destruction of the phospholipids. Figure 2 shows the SDS-PAGE of the fraction number, which is the X-axis of Figure 1. In figure 2, a constant level of protein must be used through each lane of the SDS, in every step. The sample enzyme must be …show more content…
E has the highest peak, although it's mainly protein that is not of interest to us. There are 4 bands of protein and 2 band of considerable thickness. This means there are multiple proteins of the same size present. The peak F is considerably smaller, but there seems to be more purification. There are only 3 bands of protein and on is thick. PI is also coming into play at this peak, which means the membrane of the bacteria are being affected. Peak G has even more protein purification, with only 2 bands. The thick band is around 55KD which is where phospholipase A shows to move towards. At G, protein concentration is very low, while PI and PLA are evenly high. This shows where the most activity is and where our specific enzyme is located. Moving to H, we see little to no activity and overall protein concentration seems to be increasing. Table 1 shows the multiple step purification process of phospholipase A. Homogenate had a large protein concentration, which means that our sample is far from purification. The fold purity is only 1 and bacterial activity is high.