Lab Report
Roy Levin
Bio 11 Lab
Dr.Izquierdo
Analysis of Macromolecules in Tissue Homogenates of Bos taurusMaterials and Methods
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
Bio 11 Lab
Dr.Izquierdo
Analysis of Macromolecules in Tissue Homogenates of Bos taurusMaterials and Methods
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
References: 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.