6.1 - Competent Chemist Rating
"What's in a Cow's Heart Anyway?"
- Pipetting with pipetman
- Calibrating pipetman
- Preparation of a standard curve
- Serial dilution
- UV-Vis Spectroscopy
- Discussion of Protein Assays
- Pipettmen: 100 P, 1000 P
- Pipette tips—large and small, 8 Test tubes
- Eppendorf tubes and holders
- Disposable UV-Vis cuvettes—5 mL
- You will be given a sample solution of bovine heart cytochrome c. You will use the Coomassie® Plus Protein Assay from Pierce to determine the protein concentration of the sample.
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.
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 and then empty it into a tared container. You can then obtain a weight and, 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 the 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)
- 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
||Stock - 2000 µg/mL
|375 µL of Stock
||A - 1500 µg/mL
|325 µL of Stock
||B - 1000 µg/mL
|175 µL of A
||C - 750 µg/mL
|325 µL of B
||D - 500 µg/mL
|325 µL of D
||E - 250 µg/mL
|325 µL of E
||F - 125 µg/mL
- 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.
- The Standard Protocol
- Pipette 0.05 mL of each standard or unknown sample into appropriately labeled Eppendorf tubes. Prepare 3 unknown samples.
- Use 0.05 mL of the diluent (25 mM MOPS buffer, pH 7. Provided by TA) to prepare one blank tube.
- Add 1.5 mL of the Coomassie® Plus reagent to each tube including the blank, mix well. Allow 10 minutes at RT for color to develop.
- Transfer standards, unknowns and blank to 1.5 mL UV cuvettes. Measure the absorbance at 595 nm of each tube vs. blank.
- The computer will subtract the average 595 nm reading for the blank from the 595 nm reading for each standard or unknown sample. Follow the guidelines for opening the program and operating the UV (see 9.4 UV Operation Guide).
- 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.
- Keep all of your solutions until after you have plotted and analyzed your data.
- You may need to re-do some of your UV absorptions.
- 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.
6.2 - Expert Experimentalist Rating
"A Heart as Strong as Iron"
- Disposable UV-Vis cuvettes (1-mL capacity)
- Pipettmen: 20 P, 100 P, 1000 P
- Pipette tips
- Eppendorf tubes (safe-lock)
- Boiling plate or rack to hold Eppendorf tubes
- Large crystallizing dish
- From the CC-level experiment, you should know the concentration of protein in your sample. Now you will determine the concentration of iron in bovine heart cytochrome c.
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
- 10 mM Ferrozine solution
- Saturated ammonium acetate solution
- Preparation of Standards
- Prepare a fresh set of iron standards in 2 mL Eppendorf tubes, as illustrated below. Carefully label each tube. Also fill 3 tubes with 300 µL of your unknown protein sample.
Preparation of standards.
|µL OF Fe AA STANDARD (100 µg/mL)
||µL OF BUFFER TO ADD
- Add 30 µL of ultrapure HNO3 (5 M) to each standard and sample.
- Place the closed Eppendorf tubes in a rack, and boil them for 15 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 three samples against a milli-q water blank.
- Generate a calibration curve of A562 vs. [Fe] from your standards.
- Determine the [Fe] in your unknown.
- 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.