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