WARNING NOTICE: The experiments described in these materials are potentially harzardous and require a high level ecial facilities and aquipment, and supervision by appropriate individual. You bear the sole responsibility, liability, and risk for the: Dentation of such safcty procedures and measures. MT shall have no 5.2. Expert Experimentalist Rating: "A Heart as Strong as Iron Techniques Checklist Use of a centrifuge E quipment Disposable UV-Vis cuvettes( l-mL capacity) Pipetmen: 20 P, 100 P, 1000 P Eppendorf tubes( safe-lock) Hot plate Centrifuge Boiling plate or rack to hold Eppendorf tubes Large crystallizing dish roa From the CC-level experiment, you know the concentration of protein in your sample. Now you will determine the concentration of iron in bovine heart cytochrome c Experiment Outline: The Ferrozine assay Ferrozine is an iron-chelating agent. When it forms a complex with ferrous iron (Fell), it shows a characteristic UV-Vis absorption at 562 nm. By comparing the A562 of your sample to a calibration curve of iron standards, you will determine the concentration of iron in your protein sample Solutions provided by your TA -Fe aa standard(aa= atomic absorption) Buffer -25 mM MOPS, pH 7 -Ultrapure HNO3(5 M 75 mM Ascorbic acid solution 10 mM Ferrozine solution Saturated ammonium acetate solution
5.2. Expert Experimentalist Rating: “A Heart as Strong as Iron” Techniques Checklist: • Use of a centrifuge ❑ Equipment: • Disposable UV-Vis cuvettes ( 1-mL capacity) • Pipetmen: 20 P, 100 P, 1000 P • Pipet tips • Eppendorf tubes (safe-lock) • Hot plate • Centrifuge • Boiling plate or rack to hold Eppendorf tubes • Large crystallizing dish Goals: • From the CC-level experiment, you know the concentration of protein in your sample. Now you will determine the concentration of iron in bovine heart cytochrome c. Experiment Outline: The Ferrozine Assay Ferrozine is an iron-chelating agent. When it forms a complex with ferrous iron (FeII), it shows a characteristic UV-Vis absorption at 562 nm. By comparing the A562 of your sample to a calibration curve of iron standards, you will determine the concentration of iron in your protein sample. Solutions provided by your TA: -Fe AA standard (AA = atomic absorption) -Buffer - 25 mM MOPS, pH 7 -Ultrapure HNO3 (5 M) -75 mM Ascorbic acid solution -10 mM Ferrozine solution -Saturated ammonium acetate solution 35 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. 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1. Preparation of Standards Prepare a fresh set of iron standards in 2 mL Eppendorf tubes, as illustrated below. Carefully label each tube. Also fill 2 tubes with 300 uL of your ul of fe aa standard(99 ug/mL) uL of Buffer to add 288 276 Add 30 uL of ultrapure HNO3(5 M)to each standard and sample tube Place the closed Eppendorf tubes in a rack, and boil them for 30 minutes in a hot water bath(a large Pyrex dish over a heating plate) Centrifuge for 1-2 minutes, making sure the centrifuge is properly balanced. Remove 300 uL of the supernatant liquid from each tube, and transfer to fresh tubes (labeled!) Add 1020 uL of distilled water Add 60 uL of 75 mM ascorbic acid Add 60 ul of 10 mM ferrozine Add 60 uL of saturated ammonium acetate Shake each tube and wait 10-15 minutes, the solutions should become purplish in color Transfer to a 1.5 mL cuvette, and determine the A562 for each standard and your two samples. Generate a calibration curve of A562 vs [Fe] from your standards Determine the [Fe] in your unknown. Results To obtain your EE Rating"in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.995 correlation coefficient or higher. Additionally, the bsorbance values for your unknown samples must have a standard deviation of 0.035 or less. Finally, you must determine the number of molecules of iron per molecule of protein
1. Preparation of Standards: • Prepare a fresh set of iron standards in 2 mL Eppendorf tubes, as illustrated below. Carefully label each tube. Also fill 2 tubes with 300 µL of your protein sample. µL of Fe AA standard (99 µg/mL) µL of Buffer to add 0 300 6 294 12 288 18 282 24 276 • Add 30 µL of ultrapure HNO3 (5 M) to each standard and sample tube. • Place the closed Eppendorf tubes in a rack, and boil them for 30 minutes in a hot water bath (a large Pyrex dish over a heating plate). • Centrifuge for 1-2 minutes, making sure the centrifuge is properly balanced. • Remove 300 µL of the supernatant liquid from each tube, and transfer to fresh tubes (labeled!). • Add 1020 µL of distilled water. • Add 60 µL of 75 mM ascorbic acid. • Add 60 µL of 10 mM ferrozine. • Add 60 µL of saturated ammonium acetate. • Shake each tube and wait 10-15 minutes, (the solutions should become purplish in color). • Transfer to a 1.5 mL cuvette, and determine the A562 for each standard and your two samples. • Generate a calibration curve of A562 vs. [Fe] from your standards. • Determine the [Fe] in your unknown. Results • To obtain your "EE Rating" in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.995 correlation coefficient or higher. Additionally, the absorbance values for your unknown samples must have a standard deviation of 0.035 or less. Finally, you must determine the number of molecules of iron per molecule of protein. 36