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The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present by color, was added to all samples. The spectrophotometer was zeroed at 595 nm. A standard curve was made with the different absorbencies and concentrations. After the linear equation was formed, the unknown sample concentration was determined using the standard curve equation. A Gel Electrophoresis was used to perform a qualitative analysis. The use of 5 microliters of the homogenate was heated to 80 degrees Celsius. Then the homogenate was transferred to a 2-microliter-protein gel sample buffer. Samples loaded on to the gel was run at 100 v and stained with comassie blue; observations were made next lab. (Clendening 2014)
The amount of glucose and glycerol were determined from the homogenate sample as well. Samples A(+) and B(-) were used to determine glucose. Sample A had 8mg/ml amylogloucosidase in .2M citrate buffer, pH 5.0 and was incubated at 37° Celsius for two hours to allow for the enzyme to digest the glycogen. Sample B was the control, where only .2M citrate buffer, pH 5.0 was added which was used to measure the amount of free glucose. While incubating, glycerol was determined by using 3 ml triglyceride reagent incubated at 37°celsius for 5 minutes. After warmed, 30 microliters of homogenate was added to sample. Once again, sample was incubated at 37°celsius for 10 minutes. Absorbances of glycerol standard (2.5 mg/ml) and glycerol sample with reagent were measured at 520 nm. The concentration of glycerol was determined by using this formula: (A520 homogenate/A520 of the standard) x 2.5 mg/ml glycerol. (Clendening 2014)
To find glucose and free glucose of the two samples, a standard curve was used with blank, 0.2 mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0 mg/ml glucose standards. Three milliliters of trinder reagent was first warmed at 37° celsius for 5 minutes indicating the amount of glucose by the color of the dye. Then glucose standards were added to each tube. For the blank standard, .2M-citrate buffer was only added because it was only used to calibrate spectrophotometer. All standards were incubated at 37° celsius for 10 minutes. Absorbances were measured at 520nm. A graph was drawn plotting concentration vs. absorbances and a linear equation was derived. While waiting for glycogen digestion, protein gel from the previous lab was observed and compared with bradford assay. When glycogen digestion was completed, 3 ml of trinder reagent was warmed for 5 minutes at 37° celsius. Thirty microliters of the amyloglucosidase sample was added to one sample and 30 microliters of free glucose sample was added. Once both absorbances of both samples were measured at 520nm, the concentrations of total glucose and free glucose were determined from the linear equation and multiplied by 2 to account for dilution.
Concentration of protein, DNA, and ratio of the two in heart, kidney and liver of bos taurus. Average standard deviation of protein concentration is also shown. Triglyceride concentrations in the liver are much higher than the heart and kidney. The differences between triglyceride and glycerol concentrations were triglyceride includes the free glycerol plus the glycerol. Pure glycerol concentrations were not determined because of reagent availability. The triglyceride concentrations were assumed to be proportional to glycerol concentrations. (Figure 2) -1143002095500Figure 2. The average triglyceride concentrations of heart, kidney, and liver homogenates of bos taurus are shown. Errors bars were created from 5 readings for heart, 6 readings for liver, and 7 readings for kidney. The absobances of of the enzyme-treated and free-glucose samples were measured. By using the standard curve of the glucose linear equation, the concentration of free-glucose and total glucose was found. In order to find glycogen concentrations, total glucose was subtracted from free glucose. This concentration was multiplied by two to account for the dilution. The liver had the most glycogen conentration (2.499 mg/ml) comapred to the heart(0.684 mg/ml) and kidney(0.904 mg/ml)(Figure 4)
Figure 4. Concentration of glycogen is shown in the heart, kidney, and liver homogenates of bos taurus. Errors bars were created from 5 readings for heart, 6 readings for liver, and 7 readings for kidney. Free glucose were more abundant in the liver with a concentration of 1.451 mg/ml compared to the heart (.3129 mg/ml) and kidney (.2287mg/ml). Free glucose concentrations were determined by using the non-enzyme treated sample. Using the standard curve equation, concentration was found and multiplied by 2 to account for dilution.
Figure 5. Free glucose concentrations of heart, kidney, and liver homogenates of bos taurus. Errors bars were created from 5 readings for heart, 6 readings for liver, and 7 readings for kidney. Figure 4 and 5 both indicated a similar pattern. The liver for glycogen had a concentration of 2.499 mg/ml and a free glucose concentration of 1.451 mg/ml. When compared to the heart and liver, the heart had a glycogen concentration of 0.684 mg/ml and a free glucose concentration of 0.3129 mg/ml and the kidney had 0.904 mg/ml glycogen and 0.229 mg/ml free glucose. Therefore, the liver had the highest concentration in both pools of glucose. Glycogen is a main energy storage molecule located in the liver. It is made up of glucose monomers, which are consumed by the body by eating sugars. Any excess glucose is turned into glycogen, which is stored in the liver or muscles. The heart and kidney had small amounts of glycogen, 0.684 mg/ml for the heart and 0.904 for the kidney but larger amounts of triglycerides, which were 0.7926 mg/ml for the heart, 0.806 mg/ml for kidney.
However, the liver had the highest amount of triglycerides with a concentration of 1.261 mg/ml. Triglycerides are stored in adipose cells that are abundant in all three homogenates with liver having the most of them. Some macromolecules were more abundant in different tissues than others. Figures 4 and 5 showed a larger amount of carbohydrates within the liver homogenate. Furthermore, Figure 2 indicated a greater amount of triglycerides in the liver compared to the heart and kidney. Lastly, Figure 1 and Table 1 strongly suggest a higher protein concentration in heart compared to the liver and kidney.
Clendening, B., St. Angelo, C.J., Krause, M.K, DiAngelo, J.R., and Vallier, L.G. 2014. Analysis of Macromolecules in Tissue Homogenates of Bos Taurus (Proteins, Carbohydrates, and Triglycerides). Bio 11, Biology, Hofstra University.