Introduction: The purpose of this lab is to determine the concentration of protein in liver cells using a standard curve which is made from samples containing known concentrations of Bovine Serum Albumin (BSA). We will also learn how to homogenize liver tissue samples to extract protein. The goal of this experiment is to understand the feasibility of this kind of test in determining protein concentration. This method has been used in multiple experiments including “Monitoring Oxidative Stress in Acute-on-Chronic Liver Failure by Advanced Protein Products.” (Liu, Han, Tian, Zhu, Liu, Li, Xiao, Li, Feng). In another experiment BSA protein was used as a marker for spectrophotometric determination of total protein, this means we should be able to measure changes in BSA concentration with the spectrophotometer “Spectrophotometric Determination of Total Protein in Serum Using a Novel Near-Infrared Cyanine Dye, 5.5’ –dicarboxy-1, 1’ –disulfobutyl-3,3,3’ ,3’ –tetramethylindotricarbocyanine.” (Hong, Wei-Rong, Xiao-Feng, Hua-Shan).
Hypothesis: The liver cells with more protein will yield a deeper purple color when mixed with the BCA therefor causing a higher absorption of light from the spectrophotometer.
Experimental Outline: 1. Prepare the homogenized liver sample A. Thaw a .5g liver sample and transfer to a Potter-Elvejhem homogenizer. B. Add 5ml of chilled homogenization buffer. C. Use pestle to crush (homogenize) D. Transfer mixture to a 15ml centrifuge tube E. With centrifuge set at 12,000 rpm let samples process for 15 minutes. F. Transfer the solution to a labeled cryostorage vial in a bucket of ice. 2. Determine the protein concentration of the homogenate A. Dilute a portion of homogenate 50x with the buffer to a final volume of 5ml (5000ul) 1. Homogenate needed = 5000ul / 50 = ____ ul 2. Buffer needed = 5000ul – above value = ____ ul 3. Use a 5ml pipet to measure buffer and homogenate. 4. Combine and mix the sample in a 13 mm test tube. 5. Keep on ice B. Label and Create standard curve with different BSA concentrations in 10 test tubes. 1. Label 6 tubes that will receive BSA as (0, 0.2, 0.4, 0.6, 0.8, and 1.0 ug/ul) 2. Label 4 tubes that will receive homogenate (Blank, #1, #2, and #3) 3. Add 100ul of the BSA assay samples to separate test tubes. 4. Add 100ul of diluted liver homogenate to tubes labeled #1, #2, and #3. 5. Add 100ul of homogenization buffer to tube labeled blank. 6. Add 2 ml of BCA reagent to test tubes and incubate at 45C for 15 minutes. 7. Measure absorbance of blanks and samples at 562 nm and record the data. 8. Control for the standard curve samples will be the tube containing 0 ug/ul 9. Control for the liver samples will be tube containing only homogenization buffer 10. Save tubes with 0 and .4 ug/ul for later use in at least a below freezing environment. 3. Determining how the BCA reagent reacts in the presence and absence of protein. A. Place dH20, 0 ug/ul BSA sample, and 0.4 ug/ul BSA sample into three separate test tubes. B. Negative control tubes are dH2O and 0 ug/ul BSA sample. C. Positive control tube is 0.4 ug/ul BSA sample D. Using a spectrophotometer take absorption measurements of the two BCA samples at 20nm intervals starting at 420nm and ending at 700nm. E. Zero the machine using the dH2O blank before each reading. F. Record the measurements in a table G. Take measurements in 5nm intervals at the max. H. Plot all data in excel
Materials:

References: 1. Duke. (n.d.). Spectrophotometric determination of protein concentration. Retrieved from http://www.biochem.duke.edu/wysiwyg/downloads/GrimsleyPace.pdf 2. Hong, W., Wei-Rong, L., Xiao-Feng, G., & Hua-Shan, Z. (2007). Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine. Analytical & Bioanalytical Chemistry, 387(8), 2857-2862. doi:10.1007/s00216-007-1153-y

3. Marietta. (n.d.). Measuring protein concentration through absorption spectrophotometry. Retrieved from http://www.marietta.edu/~spilatrs/biol309/labexercises/Spectrophotometry.pdf 4. Sayeed, M., Habib, R., Rahman, M., Banna, H., & Rana, S. (2012). Interaction of Ketotifen Fumarate with Anhydrous Theophylline in Simulated Gastric and Intestinal Media and Effect on Protein Binding. Tropical Journal Of Pharmaceutical Research, 11(2), 263-268.