Lab 2-3 Lab Report
Analysis of Protein, Carbohydrates, and Triglycerides in Bos taurus Tissue I) Materials and Methods:
Homogenates were provided made from liver, kidney, or heart in a 1:20 ratio with sucrose-phosphate buffer that was stored at -70° C. The tissue I tested was the liver homogenate. To qualitatively analyze proteins of the homogenate, 5µl of original liver homogenate was combined with 2 µl of protein gel sample buffer and heated in a water bath at 80° C for 10 minutes. After centrifuging the sample for 5 seconds, the contents were loaded onto a polyacrylamide protein gel set at 100V for Electrophoresis and stained Coomassie blue for an hour and results were observed the next class period a week later (Clendening). To determine Protein Concentration of the homogenate, using a 1:5 ratio, 50 µl of original homogenate was diluted in 200 µl of water and labeled dilute homogenate. 30 µl measurements of 0.4, 0.8, 1.2, 1.6 and 20 mg/ml were added into their own sample of the 3 ml Bradford Assay, 30 µl of water was added to a separate sample of Bradford Assay and 30 µl of the dilute liver homogenate was added to its own sample of Bradford Assay and left for 3 minutes. The absorbance of the 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 (Clendening). Y= 0.52x + 0.186 , R2= 0.9572
Protein to DNA ratio was calculated by dividing the determined average protein concentration of the homogenate by the given DNA concentration for all three Bos taurus homogenates (Clendening).Original homogenates were used to digest Glycogen. First, 50 µl of 8 mg/ml amyloglucosidase was added to 50 µl of homogenate to create a reaction to digest glycogen. Another 50 µl of homogenate was then combined with 50 µl of 0.2 M citrate buffer to act as a control with no enzyme to digest glycogen. The reactions were incubated at 37°C...
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