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After centrifuging the sample for 5 seconds, the contents were loaded onto a polysaccharide protein gel set t IV for Electrophoresis and stained Accompanies blue for an hour and results were observed the next class period a week later (Gladdening). To determine Protein Concentration of the homogenate, using a ratio, 50 Pl of original homogenate was diluted in 200 Pl of water and labeled dilute homogenate. 30 Pl measurements of 0. 4, 0. 8, 1 . 2, 1. And 20 MGM/ml were added into their own sample of the 3 ml Bradford Assay, 30 of water was added to a separate sample of Bradford Assay and 30 Pl of the dilute liver homogenate was added to its own sample of Bradford Assay and left for 3 minutes. The absorbency of he standards and the diluted homogenate samples were recorded to create a standard curve. From this equation, the protein concentration of my sample was determined. Class averages were also obtained (Gladdening). Y? 0. Xx + 0. 186 , 0. 572 Protein to DNA ratio was calculated by dividing the determined average protein concentration of the homogenate by the given DNA concentration for all three Boss Taurus homogenates (Gladdening). Original homogenates were used to digest Glycogen. First, 50 Pl of 8 MGM/ml maliciousness’s was added to 50 Pl of homogenate to create a reaction to digest glycogen. Another 50 VI f homogenate was then combined with 50 Pl of 0. 2 M citrate buffer to act as a control with no enzyme to digest glycogen. The reactions were incubated at 37 co for 2 hours and we later observed absorbency (Gladdening).
During incubation, homogenate triglycerides were measured by assuming that triglycerides in all homogenates were equal to total glycerol. Glycerol standard sample was given to the class. One sample of 3 ml Triglyceride reagent was heated at 37 co for 5 minutes, then mixed with 30 Pl of Boss Taurus homogenate and incubated for 10 more minutes at the same temperature. Absorbency of the glycerol standard ND homogenates were measured, and converted to concentration of glycerol (Gladdening). ( AWAY homogenate / AWAY of the standard ) x 2. MGM/ml glycerol The average triglyceride amount for each homogenate was determined. To make a standard curve of glucose, 6 samples of 3 ml of Trendier reagent were heated at ICC. After 5 minutes, 30 Pl of 0. 2 M citrate buffer was added to one sample, 30 Pl of 0. 2, 0. 4, 0. 6, 0. 8, and 1. 0 were added to one of the remaining 5 samples of Trendier reagent and heated for 10 minutes at the same temperature. Absorbency of the standards was measured (Gladdening). Y= . Xx – . 0005, RE = . 871 To measure the free glucose and glycogen, 2 samples of 3 ml of Trendier reagent were warmed at 37 Co.
After 5 minutes, from the digestion of glycogen process, 30 Pl of the maliciousness’s-treated reaction was added to one sample and 30 Pl of the free glucose control was added to the other sample. The samples were heated for 10 minutes at ICC and absorbency was recorded. Concentration of free glucose was calculated when solving for x using the absorbency of the reaction without maliciousness’s with the above equation. The same was done to find concentration of total glucose using the absorbency of the reaction with milliseconds. Total glycogen was then calculated by subtracting free glucose from total glucose (Gladdening).
II) Results: Kodak. 250 150 100 75 50 37 25 20 15 10 Figure 1. Protein electrophoresis of Boss Taurus Heart, Kidney and Liver Homogenates. Lane 1 is the protein ladder in Kodak. The heart homogenate is located in lanes 2-5, kidney homogenate in lanes 6-10, and liver homogenate in lanes 11-15. Table 1. Protein and DNA concentrations, and Protein to DNA ratio of Boss Taurus heart, kidney and liver homogenates. Protein Con. Based on class averages. DNA Con. Ratio Heart n=17 4. 53 MGM/ml 1. 05 peg/ ml 4. 1 MGM protein / peg DNA Kidney n=15 4. 97 MGM/ml 5. 79 peg/ ml 0. 86 MGM protein / peg DNA Liver n=14 2. 5 MGM/ml 7. 64 peg/ ml 0. 39 MGM protein / peg DNA Figure 2. Total triglyceride concentration (MGM/ml) in the Liver, Kidney, and Heart homogenates. Class averages shown. Liver n=12, Kidney n=11, Heart n=16. Figure 3: Total glycogen concentration (MGM/ml) in the Liver, Kidney, and Heart homogenates. Class averages shown. Liver n=18, Kidney n=14, Heart n=14. 228600133985 Figure 4: Total free glucose concentration (MGM/ml) in the Liver, Kidney, and Heart Looking at Protein Concentration (Figure 1), the homogenate tissue with the cost abundant concentration was the heart.
It shows the most pronounced bands of proteins, especially the 250 Kodak band, as well as the greatest amount of visible bands of proteins. The difference in concentration between Kidneys and Liver homogenates is more difficult to differentiate. Contrastingly, Table 1, which is based on class averages, shows that the homogenate with the highest protein concentration is the kidney as opposed to the heart. Table 1 also shows Protein/DNA ratio, where the heart is the highest homogenate. We can infer that the large amount of protein per DNA could mean that the cells must be larger o accommodate for the abundance of macromolecule.
Triglycerides (Figure 2) of each homogenate are a reflection of the amount of glycerol because the absorbency readings of glycerol were converted to triglyceride concentration during the experiment. Here we see that the kidney homogenate had the highest concentration of triglycerides followed closely by the liver homogenate. In order to obtain values for glycogen, free glucose and total glucose concentrations had to be calculated using the glucose standard curve. Once these numbers were known, subtracting free glucose from total glucose came out to the amount of logger.
Figure 4 shows free glucose levels, where the liver had the highest amount and the kidney the lowest. Figure 3 provides glycogen concentration, where again the liver has the highest levels, but the heart homogenate has the lowest. To conclude, each cell type of the Boss Taurus had varying levels of macromolecules according to the experiments performed in these labs. Reference: Gladdening, B. , SST. Angelo, C. J. , Krause, M. K. , Tangelo, J. R. , and Valier, L. G. 2014. [Analysis of Macromolecules in Tissue Homogenates of Boss Taurus Apart and Apart]. Bio 11, Biology, Hoofers University.