The experiments described in these materials are potentially hazardous and require ahigh lovel al facilitics and equip ment, and supervision by appropriate individual. You bear the sole and risk for the implementation of such safty procedures and measures. MTT shall have no or risk for the content or implemcatation of amy of the material presented. Legal Notices 5. Protein Assays and Error analysis 5.1. Competent Chemist Rating:"What's in a Cow's Heart Anyway? Techniques Checklist: Pipetting with pipetman Calibrating pipetman Preparation of a standard curve Serial dilution UV-Vis Spectroscopy Pre-Lab Discussion of Protein Assays E Pipetman: 100 P, 1000 P Pipet tips-large and small 8 Test tubes Eppendorf tubes and holder Disposable UV-Vis cuvettes-5 mL Goal: You will be given a sample of a solution of bovine heart cytochrome c. You will use the Coomassie( Plus Protein assay from Pierce to determine the concentration of tein in the sample Note You will receive a tray of Eppendorf tubes: one containing stock solution, three containing 50 uL each of bovine heart cytochrome c, and several empty tubes for mixing solutions. You will also be provided with a bottle of 25mM MOPS buffer, pH7
5. Protein Assays and Error Analysis 5.1. Competent Chemist Rating: “What's in a Cow's Heart Anyway?” Techniques Checklist: • Pipetting with pipetman ❑ • Calibrating pipetman ❑ • Preparation of a standard curve ❑ • Serial dilution ❑ • UV-Vis Spectroscopy ❑ Pre-Lab: • Discussion of Protein Assays Equipment: • Pipetman: 100 P, 1000 P • Pipet tips - large and small • 8 Test tubes • Eppendorf tubes and holder • Disposable UV-Vis cuvettes - 5 mL Goal: • You will be given a sample of a solution of bovine heart cytochrome c. You will use the Coomassie® Plus Protein Assay from Pierce to determine the concentration of protein in the sample. Note: • You will receive a tray of Eppendorf tubes: one containing stock solution, three containing 50 µL each of bovine heart cytochrome c, and several empty tubes for mixing solutions. You will also be provided with a bottle of 25mM MOPS buffer, pH 7. 32 WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level ofsafety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. Legal Notices
Experiment Outline: Pipetman calibration Prior to beginning any experiment with a pipetman, it is necessary to first calibrate it. This procedure will determine exactly how much liquid is delivered when a certain amount is "dialed-in to the instrument. To calibrate your pipetman, simply draw up a certain amount of water, empty it into a tared container, and obtain a weight. Knowing that water has a density of 1.00 g/mL, you can perform a calculation to tell you the accuracy of your pipetman. Most instruments will need no correction,and ones that are incorrect will usually be off by no more than I PL The coomassie@-Protein Reaction Scheme This protein assay works by forming a complex between the protein and the coomassie dye. When bound to the protein, the absorbance of the dye shifts from 465 nm to 595 nm(A595) You will first generate a standard curve using the protein Bovine Serum Albumin (bsa)by measuring the absorbance at 595 nm of a series of standards of known concentration. Next, you will measure the A595 of your sample and determine its concentration by comparison to the standard curve Protein CoomassieG-250 in acidic medium---> Protein-Dye complex(blue; measured at 595 nm) 1. Preparation of diluted bsa standards Prepare a fresh set of protein standards by diluting the 2.0 mg/mL BSA stock standard (Stock) as illustrated below. There will be sufficient volume for three replications of each diluted BSA standard, if necessary Vol of the bSa to Add Vol of Diluent(buffer) to Add Final BSA Conc. 300 uL of Stock OuL Stock-2000 ug/mL 375 uL of Stock A-1500 ug/L 325 uL of Stock B-1000 ug/mL 175 uL of A 175 C-750 ug/mL 325 uL of 325uL D-500 ug/mL 325 uL of D 325uL E-250 ug/mL 325 uL of E 325比L F-125 ug/mL 100 uL of 400uL s ug/m
Experiment Outline: Pipetman Calibration Prior to beginning any experiment with a pipetman, it is necessary to first calibrate it. This procedure will determine exactly how much liquid is delivered when a certain amount is "dialed-in" to the instrument. To calibrate your pipetman, simply draw up a certain amount of water, empty it into a tared container, and obtain a weight. Knowing that water has a density of 1.00 g/mL, you can perform a calculation to tell you the accuracy of your pipetman. Most instruments will need no correction, and ones that are incorrect will usually be off by no more than 1 µL. The Coomassie®-Protein Reaction Scheme This protein assay works by forming a complex between the protein and the Coomassie® dye. When bound to the protein, the absorbance of the dye shifts from 465 nm to 595 nm (A595). You will first generate a standard curve using the protein Bovine Serum Albumin (BSA) by measuring the absorbance at 595 nm of a series of standards of known concentration. Next, you will measure the A595 of your sample and determine its concentration by comparison to the standard curve. Protein + Coomassie®G-250 in acidic medium---> Protein-Dye complex (blue; measured at 595 nm) 1. Preparation of diluted BSA standards • Prepare a fresh set of protein standards by diluting the 2.0 mg/mL BSA stock standard (Stock) as illustrated below. There will be sufficient volume for three replications of each diluted BSA standard, if necessary. Vol of the BSA to Add Vol of Diluent (buffer) to Add Final BSA Conc. 300 µL of Stock 0 µL Stock - 2000 µg/mL 375 µL of Stock 125 µL A - 1500 µg/mL 325 µL of Stock 325 µL B - 1000 µg/mL 175 µL of A 175 µL C - 750 µg/mL 325 µL of B 325 µL D - 500 µg/mL 325 µL of D 325 µL E - 250 µg/mL 325 µL of E 325 µL F - 125 µg/mL 100 µL of F 400 µL G - 25 µg/mL 33
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 protein
2. 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
