We can see in the above graph that the Bovine Insulin at 65 degrees varies depending on the pH level of the solution. Looking closely to the result, using pH 2.0 intensifies Th T fluorescence Intensity from one day to one week but there is a sudden drop on the second week. It is almost the same phenomenon to solution using pH 4.0 solutions. But looking on the pH 7.0, it is not the same intensity to pH 2.0 though, but up to the second week, the behavior of bovine insulin does change gradually, unlike to those in pH 2.0 and pH 4.0.However, on the second graph that shows the behavior of Bovine Insulin at 37 degrees, we can see that it is not as intensified as in the 65 degrees but in pH 2.0 the behavior is the same but in pH 4.0, the behavior somehow defy from that observed in 65 degrees. Noticeably, in pH 7.0, there is no Th T fluorescence intensity observed.
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Comparing the result of pH 7.0 under 37 degrees and 65 degrees, we can see that the existence of bovine insulin under certain controlled condition. The result of this test was confirmed by examining series of samples under electronic microscope that has shown fiber formation in the form of thin and long fibers which are not branched for pH 2.0, pH 4.0 and pH 7.0 at 65 degrees. But, observing the same condition but modifying the temperature which is 37 degrees, pH 7.0 did not show any fiber formation but only showed amorphous aggregate. The negative control was monomeric insulin protein for each pH without any incubation and supposed not to give any sign or signal. But since it is incubated, there are observable traits that can be seen.
In these two graphs, we can see the effects of seeding to the Th T fluorescence intensity of Bovine insulin. Under 65 centigrade, we can see that still, pH 2.0 performed the highest intensity among the group and from the beginning, it created a gap to the other level of pH. It is observable among the three pH levels that the longer the time consumed, the more intensified it gets. In pH 2.0 , it reached its plateaus level later on the day two and it has a consistent intensity until the last day of observation. However, under the same conditions, Bovine Insulin Th T fluorescence intensity pH 4.0 and pH 7.0 created the same activity with regards to pH 2.0. but the intensity is not that high compared to the latter. Another thing is it reached its plateau stage earlier than pH 2.0 and close observation will reveal that pH 4.0 and pH 7.0 started to act in the same intensity when they reached their plateau level. Comparing them to pH 2.0 that showed higher intensity in that stage, it is very apparent that since they (pH 4.0 and pH 7.0) reached their plateau level earlier, pH 2.0 continued to intensified it Th T fluorescence.
Comparing the result on the Bovine insulin with seeding under the same temperature, it is apparent that it has more intensity that the result without seeding. Observing their behavior, they are somehow closer in this condition at the end than the start in contrast in the phenomenon they showed without seeding. They also somehow reached their plateau stage earlier (within five hours). Although, observing closely the result, they are quite unstable in their plateau stage but nevertheless they exhibit closer intensity.
In this graph, we can see that Na2SO4 has certain effects on the behavior of Bovine insulin under 65 centigrade and 37 centigrade. Under the first temperature, pH 2.0 exhibit unexpected result on the first day which is lower intensity without control. This is the exact opposite of the solutions and observations under pH 4.o and pH 7.0 which is consistent through out the period of observation. They showed higher intensity if the control was added to them. And the change is very much apparent and observable through out time unlike pH 2.0. But, one the next days, pH 2.0 showed higher intensity with controls.
Under 37 centigrade however, we can see that pH 2.0 reacted as expected which showed sudden change in intensity (for pH 4.0). but an interesting part of it is that pH 4.0 almost showed the same intensity when the control is added to it. This is noticeable through out the experimentation days. Although in pH 7.0, there is no apparent observable result, EM Examination revealed curly fibers for pH 7.0
The result of the experiment was gathered through subjecting each Bovine insulin sample to a series of tests by incubating some samples without Na2SO4 which is referred to as controlled environment and comparing the result of the observation to that of with Na2SO4.
The graphs showed the effects of glucose on Bovine insulin fibrillation. There are four conditions set to be able to attain the expected result. On the first condition, there is 8.5 mM glucose at 65 degrees centigrade. It is important to remember that the controlled condition is used to describe solutions without glucose at all. On the first day, the observation was all levels of pH showed the same effect which is decreased intensity suddenly. On the second they however, only pH 4.0 and pH 7.0 showed sudden decrease compared to pH 2.0 which is a little decrease only. But surprisingly, on the third day, all of them showed sudden decrease except for pH 7.0 which showed almost the same intensity level.
Changing the concentration of glucose into a higher dose, the effect of the process is almost the same with compared to the first one. From day one to day three, all pH levels showed sudden decrease on their intensity. But looking on the longer run, which is in one week, the same phenomenon happened to pH 2.0 as that which happened on the first situation. There is an decrease in intensity in contrast with pH 4.0 and pH 7.0 which showed increase.
On the third situation, the concentration of glucose is 8.5 mM but the temperature is only 37 degrees. We can see that throughout the time span of the experiment, the behavior of Th H fluorescence intensity of Bovine insulin increased as compared with controlled situation. This phenomenon is apparent through out pH levels and time. But an interesting situation is that the controlled situation showed almost the same level of pH 2.0 and pH 4.0 in controlled situation.
Lastly, the concentration of glucose is higher than the previous and the same process was done. Observing the effect, it does not deviate from the phenomenon observed in the previous one. There is a sudden increase in the intensity of the solution as compared to the controlled situation. This is observable through out the levels of pH.
Comparing the result of the two different concentration across different temperature. In general, the pattern of decrease of intensity is observable in higher concentration of glucose in the two temperature. Although, some variation showed different observable condition.
This graph showed the effect of different concentration of Aceto Acetic Acid when incubated with Bovine insulin under different temperature. The activity of each insulin was observed closely monitor their activity across different times.
The first graph showed the result of the activity of Bovine insulin with 0.5 mM Aceto Acetic Acid at 65 degrees centigrade. The general trend of observation is that there is a increase in terms of intensity as compared to controlled situation which referred to as the situation without any traces of Aceto Acetic Acid incubated in the same temperature. It is really noticeable as compared to different levels of pH. But, an interesting trait of this situation is that pH 4.0 and pH 7.0 levels of solution showed a sudden increase of intensity across time but in contrast to pH 2.0 which showed sudden increase on the first day but the increase on the following days are gradual only.
Almost the same trend happened on the second situation wherein the temperature is the same but the concentration of the Aceto Acetic Acid is changed. The concentration now is higher which is 1.5 mM. the trend is basically the same. There is a increase in the intensity of the RFU across different levels and time and basically, the same phenomenon happened on pH 2.0. However, there is an interesting observation in the behavior of pH 7.0 solution in which it showed almost the same intensity in 3 days and one week as compared to pH 2.0 with Aceto Acetic Acid.
Using the two concentration in 37 degrees centigrade, there are different trend that can be observed. With regards to pH 2.0, on the first day, there is a sudden increase as compared to the controlled situation. But on three days up to one week, there is a sudden decrease on its intensity. On the other hand, the behavior of the pH 4.0 and pH 7.0 is still the same as the previous observations in the previous sets. There is a sudden decrease on the intensity of Bovine insulin RFU.
On the last concentration however, there are still different trends that showed by different levels of pH. With respect to pH 2.0. on the first day, there is an increase in RFU. But on the loner run, there is an observable decrease in the solution. However on pH 4.0 and pH 7.0, there is always an increase through out the time span. An interesting observation is the behavior of the bovine insulin with 1.5 mM Aceto Acetic Acid at 37 centigrade. The intensity is somehow reversed, meaning, the more acidic, the lesser intensity.
The graphs showed the effects of acetone to bovine insulin. Comparing the controlled situation, which does not contain any acetone with solutions under 65 degrees centigrade with 0.5 mM acetone, the trend is generally increase in RFU.On the other hand, higher concentration of acetone produced higher RFU in the same temperature condition across different pH levels and times. But still, the trend is generally the same for all of the setting done to observe their activities.Under lower temperature with 0.5 mM acetone, pH 2.0 showed increase on the first day but on the third and first week, its trend became decreasing. On the other pH levels however, the trend is generally increasing the level of their RFU across different time span.
Considering the same temperature used but with higher concentration of acetone which is 1.5 mM, the trend for pH 2.0 is still the same, increase on the first day but on the third day and on the first week, they decreased in the same manner as the previous situation. Overall, the higher concentration used to define RFU in Bovine Insulin, the higher the RFU you will get as compared with the controlled one.
The graphs showed the intensity of human insulin under two different temperature. On the first graph, under 65 degrees centigrade, the intensity of human insulin is increasing from the first day until the first week but suddenly dropped at the second week. The trend is still the same, the more acidic it is, the higher intensity it will be.
Under 37 degrees centigrade however, there is no observed activity for pH 7.0 as compared to other pH levels but still, the intensity is way lower than the higher temperature. The observation is still the same. The more acidic it is, the higher intensity and from the first day until the first week, the intensity is increasing but on the second week. It suddenly dropped.
The graphs showed the effects of seeding in human insulin under two different temperature. Under 65 degrees centigrade, the difference between the three pH levels are not that visible until the plateau stage in which it is quite noticeable that they almost have the same behavior.
However, under 37 degrees centigrade, pH 2.0 had set a bigger gap at the start but suddenly established an stable and narrower gap as time passed by. They almost reached the plateau stage at the same time.The graphs showed the behavior of human insulin with Na2SO4 as compared to human insulin without Na2SO4 dubbed as controlled situation. Under 65 degrees centigrade, pH 2.0 showed increase in RFU on the whole duration although it is not that significant on the 3rd day and the first week. However, in pH 4.0 and pH 7.0, the behavior is the opposite which is a decrease in RFU.
Under 37 degrees centigrade the there is a decrease in RFU as noticed across different pH levels and across different times. Although, it is apparent in pH 2.0 that the difference is not that big.The graphs showed the effect of different concentration of glucose incubated in different temperature. Under 65 degrees centigrade with 8.5 mM glucose as compared with the result of the controlled situation without glucose at all, pH 2.0 showed increase in RFU on the first day and then it suddenly showed decrease later on. It is the same phenomenon that happened on pH 4.0 and 7.0.Under the same temperature but with higher glucose concentration, the intensity as a whole is higher than the same temperature condition but with lower concentration. The trend is still the same, on the first day, pH 2.0 showed increase in RFU then suddenly decreased on the later days. Meanwhile, on the other pH levels, the trend is the same with pH 2.0In a lower temperature at 37 degrees centigrade with 8.5 mM glucose, the trend is increase of RFU throughout different levels of pH and different time span. But on the first week, there is a decrease on RFU both in pH 2.0 and pH 4.0 but increase on pH 7.0.
Increasing concentration of glucose which is 16 mM under the same temperature, as compared with the lower concentration under the same temperature, it is quite higher but it is not that significant. The trend is still the same as the previous one. There is an increase in RFU across different levels and span of time except for the first week where there is a decrease on pH 2.0 and pH 4.0.
3.9 The graph showed the effect of aceto acetic acid to human insulin under different temperature. Comparing the result of the controlled situation which does not contain aceto acetic acid in the non-controlled one which used 0.5 aceto acetic acid under 65 degrees centigrade, showed that there is no activity on day one on non contolled one at pH 4.0 and pH 7.0. the general pattern across different levels is decrease in RFU but noticeably, comparing pH 4.0 and pH 7.0, the latter showed more fibrillation as compared to the previous one.
Subjecting the solutions on higher concentration of aceto acidic acid, generally, it produced higher RFU than the lower concentration. But the behavior of pH 4.0 is different from the other two wherein it showed consistent decrease through out the time span whereas the other two imposed decrease on the first day but sudden increase on the latter days.Subjecting the solutions on lower temperature but with 0.5 aceto acidic acid, it is noticeable on the first day that there is no activity for more acidic pH level under uncontrolled condition. While the general trend of all of the two acidic state is actually decrease in RFU. But noticeable, pH 7.0 reacted higher RFU and somehow, the expected result was reversed wherein the lower the acidity, the higher the RFU.
Still under the same temperature but with 1.5 mM aceto acetic acid, in comparison with the previous one, it showed higher RFU across different pH levels and time span. The overall trend of all the pH levels under different time span is increasing.The graphs shows the effects of acetone on human insulin fibrillation under different temperature. Subjecting human insulin under 65 degrees centigrade with out acetone referred to as controlled situation, it is increasing on the first day as compared to uncontrolled one, with 0.5 mM of Acetone contained on it, but on the latter part of the time span, the trend suddenly changed.
Subjecting under the same temperature with higher dose of acetone at 1.5 mM gives off higher RFU level as compared to lower concentration of acetone. But the trend is basically the same, it increased on the first day and decreased on the latter part of the experiment.On the other hand wherein the temperature is lower, under 0.5 mM of Acetone, the trend is actually increasing in all pH levels across different time span which is the same condition under the same temperature with 1.5 mM of acetone but still, the higher the acetone concentration, the higher the RFU.
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