Bio 11 Lab
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.