2. Mixing of the Coomassie@B Plus Protein Assay Reagent Allow the Coomassie( Plus reagent to come to room temperature. Mix the Coomassie@ Plus reagent solution just prior to use by gently inverting the bottle several times Do not shake 3. The standard Protocol Pipet 0.05 mL of each standard solution into appropriately labeled Eppendorf tubes. Prepare at least three samples of your unknown solution. Use 0.05 mL of the diluent(25 mM MOPS buffer, pH7, provided by ta)to prepare two blank tubes Add 1.5 mL of the Coomassie(R Plus reagent to each tube, mix well. Measure the absorbance at 595 nm of each tube versus a water reference Subtract the average 595 nm reading for the blanks from the 595 nm reading for each standard or unknown sample Prepare a standard curve by plotting the average blank corrected 595 nm reading for each BSA standard versus its concentration in ug/mL. USing the standard curve determine the protein concentration for each unknown samp Helpful Hints Keep all of your solutions until after you have plotted and analyzed your data You may need to redo some of your UV absorptions Results: To obtain your "CC Rating"in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.930 correlation coefficient(r value) or higher Additionally, the results from your absorbance values of the unknown should have a standard deviation of less than 0.048. Finally, you must determine the concentration of your unknown protein2. Mixing of the Coomassie® Plus Protein Assay Reagent: • Allow the Coomassie® Plus reagent to come to room temperature. Mix the Coomassie® Plus reagent solution just prior to use by gently inverting the bottle several times. Do not shake. 3. The Standard Protocol • Pipet 0.05 mL of each standard solution into appropriately labeled Eppendorf tubes. Prepare at least three samples of your unknown solution. Use 0.05 mL of the diluent (25 mM MOPS buffer, pH 7, provided by TA) to prepare two blank tubes. • Add 1.5 mL of the Coomassie® Plus reagent to each tube, mix well. • Measure the absorbance at 595 nm of each tube versus a water reference. • Subtract the average 595 nm reading for the blanks from the 595 nm reading for each standard or unknown sample. • Prepare a standard curve by plotting the average blank corrected 595 nm reading for each BSA standard versus its concentration in µg/mL. Using the standard curve, determine the protein concentration for each unknown sample. Helpful Hints: • Keep all of your solutions until after you have plotted and analyzed your data. You may need to redo some of your UV absorptions. Results: • To obtain your "CC Rating" in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.930 correlation coefficient (R value) or higher. Additionally, the results from your absorbance values of the unknown should have a standard deviation of less than 0.048. Finally, you must determine the concentration of your unknown protein. 34