The crude extract was retrieved by homogenizing the bovine muscle tissue. The total enzyme in the muscle tissue can be illustrated through the total activity present in the crude extract, which was 5500 ± 200 units. Next the 40% ammonium sulfate step was performed. Ammonium sulfate was slowly added to a beaker that contained the crude extract until a 40% saturation level was reached. After centrifugation the pellet (which contained low soluble proteins and other cellular debris) was discarded and the supernatant was kept. The total protein was 124 ± 6 mg and the specific activity was 4500 ± 300 units. This resulted in a specific activity of 36 ± 4 units/mg. The yield was calculated to be 82 ± 9% and the purification factor was 1.4 ± 0.3. There was not much of an increase in the purity, but there was a decrease in the total protein and the specific activity as compared to the crude extract which suggests that that unwanted protein was removed during this purification step.
Performing the 60% ammonium sulfate step further purified the 40% supernatant. The ammonium sulfate and was added to the 40% supernatant until a 60% saturation level was reached. This was then centrifuged, but this time the pellet was kept while the supernatant (which contained material such as nucleic acids and sugars) was discarded. The total protein recovered was 56.4 ± 0.4 mg and the total activity was 4140 ± 50 units. This produced a specific activity of 73 ± 1 units/mg. The yield was calculated to be 76 ± 4% and the purification factor was 2.8 ± 0.3. Each of these steps was consistent with the trends present in the theoretical values data because the purity increased. This indicates that the unwanted material was removed so that the 60% pellet can be us...
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...olecular weight). By using the protein concentration and enzyme concentration of E10 and E11 the specific activity values can be calculated. The specific activity of E10 is 497.68 units/mg and E11 is 516.6 units/mg. The m form is assumed to have a lower binding affinity (lower Km) so this would elute out first thus we can conclude that the m form is present in the E10 fraction and the h form is present in the E11 fraction.
The isozymes are assumed to have the same molecular weight but varying charge strength. With this information an ion exchange column can be performed in order to help separate the isozymes. For an anion exchange column the beads would be positive. By making one of the isozymes more negative it would bind to the beads while the other isozyme would elute out. A gel column can then be performed to confirm the presence of different isozymes.
Given that the shapes of the peaks are not ideal, they are different from the percentage height for each component. As such, the area measurement is used, to determine the concentration. For the first peak, the percent area of total was 49.65% and for the second peak, the percent area of the total was 50.35%, which reflects the quantity of the analyte that exists in the chromatogram. The retention time in both cases is constant and the pattern of peaks is constant which reflects that the conditions of testing are constant. Based on the percentages it can be seen that the major product was 2-isopropyl-1,4-dimethylbenzene at 50.35% and the minor product was 1,4-Dimethyl-2-propylbenzene at 49.65%. Therefore, on Figures 2 and 3 the peak at 2.93 is the major peak and it represents 2-isopropyl-1,4-dimethylbenzene and the peak at 2.58 is the minor peak and represents
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
a fair test, the free enzyme must be diluted to the same extent as the
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
In this experiment, in the first part, the best concentration of enzyme was determined by recording the absorption over time. In the second part, the best concentration was selected from the previous experiment which was C and the optimum pH was determined.
The major sites for the production of ammonia are the intestines, liver, and kidneys. It is biosynthesized through normal amino acid metabolism. The kidneys generate ammonia from glutamine by the actions of renal glutaminase and glutamate dehydrogenase. Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine, which is absorbed from the intestine by the portal vein. Amines obtained from diet and monoamines that serve as neurotransmitters or hormones can create ammonia by action of amine oxidase. In purine and pyrimidine catabolism, amino groups attached to the rings are released as ammonia.
A propellant is used in the military application and it is consists of oxidizer, fuel, plasticizer, curing agent and cross linkers. The commonly used oxidizer is ammonium perchlorate. But its combustion products are environmentally hazardous. Nowadays, ammonium nitrate is gaining great attraction in the field of propellant as a great oxidizer. Ammonium nitrate is an inorganic compound used as fertilizer, oxidizer and also for other applications. AN combined with fuel can be used in gas generators . The ammonium nitrate extracted from demilitarized rocket motors can be used as an oxidizing agent in the hydrothermal oxidation of organic wastes . It has seven phase transitions3.
Materials and Methods: An ion exchange chromatography column was obtained and set up for purification with the addition of 0.5 ml ion exchange matrix. 1 ml
Redwan, El-Rashdy M. "Animal-Derived Pharmaceutical Proteins." Journal of Immunoassay and Immunochemistry 30.3 (2009): 262-90. Web.
Meaning for 50ml, 0.4 grams of agarose was weighed on a scale. And with a 250ml flask, 50ml of 1X TBE buffer was added as well as the 0.4 grams of agarose (Upadhyaya, 2017). Then plastic was placed over the flask and put in the microwave for one minute. After safely retrieving the hot flask from the microwave with a mitten, it was observed to ensure that the agarose was completely dissolved and was placed on a table to cool down to not burn the casting tray; once cooled 2.5µL of ethidium bromide was added to the agarose solution, which was used to illuminate the gel once placed under UV light (Upadhyaya, 2017). Before pouring the solution into the casting tray, a comb was placed on the middle of the edge of the tray to create wells once the gel was solid, allowing for multiple DNA samples to be used at the same time to be compared against each other. After the gel was solidified, the tray was repositioned as the positive pole was closer to the wells which then would not show much results. After adding 250ml of 1X TBE buffer to the tray, an almost equal amount on both sides, submerging the gel, the comb was removed and the wells were present (Upadhyaya,
Moreover, the class average curve shows a similar trend, as the curve flattens, at 70% but with an enzyme activity of 5.3 x10-3 seconds. This indicates that even though the saturation point is the same it was considerably lower than our results, which could indicate sources of systematic error in the design of the practical.
Thus, this study will put an efforts by producing plant-derived protein to decrease the global demands for FBS and to decrease the number of bovine fetuses required for the production of FBS. It is crucial to conduct a study that is not only able to produce protein hydrolysates from tempeh, but also to increase the growth performance of cells when tempeh hydrolysates is tested on cell culture. It is significant since the tempeh hydrolysates can be a good substitute for animal-derived serum such as fetal bovine serum because it is more ethical, cost-effective and environmental friendliness. In this experiment, tempeh will undergo solvent extraction to produce Then, the protein isolate will be enzymatically hydrolyse to produce peptides. Lastly, tempeh hydrolysate will be tested on human skin fibroblast cell culture and the growth
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.
Due to the nature of amino acids, a titration curve can be employed to identify