20.109(S17):Evaluation of purified protein (Day3)
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Introduction
Protocols
Part 1: Visualize purified protein with polyacrylamide gel electrophoresis
- Last time you prepared cell-normalized -IPTG and +IPTG samples and added Laemmli sample buffer (containing SDS, etc) to them. You also have aliquots of your purified wild-type and X#Z mutant IPC proteins. Now you will complete protein denaturing in preparation for PAGE, alongside two different MW ladders.
- The pre-stained ladder will be used to track gel progress.
- The unstained ladder contains a known amount of protein per band and thus can be used to estimate gross protein contents.
- Boil all 6 samples for 5 minutes in the water bath located in the fume hood.
- Be sure to put cap-locks on the eppendorf tubes that contain your samples to ensure the lids do not open during boiling.
- You will be shown by the teaching faculty how to load your samples into the SDS-PAGE gel. You should load your samples according instructions below, one team per gel.
- Note the starting and stopping time of electrophoresis, which will be initiated by the teaching faculty at 200 V, and run for 30-45 minutes.
- Pry apart the plates using a spatula, and carefully transfer your gel to a staining box.
- Add enough distilled water to cover the gel (~200 mL) and rinse the gel for 5 minutes on the shaker.
- Repeat the rinse two more times with fresh water (~200 mL and 5-minute incubation each time).
- Add 50 mL of BioSafe Coomassie, and incubate for at least 1 hour.
- Empty the staining solution into the waste container in the fume hood - careful not to lose your gel!
- Add 200 mL of water to your stained gel. Replace with fresh water just before leaving the lab if you have a chance.
- Tomorrow, the teaching staff will transfer each gel to fresh water, then photograph them and post the results to the Day 7 Discussion page.
Part 2: Measure protein concentration
Part 2a: Prepare diluted albumin (BSA) standards
- Obtain a 0.25 mL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of diH2O from the front bench.
- Prepare your standards according to the table below using dH2O as the diluent:
- Be sure to use 5 mL polystyrene tubes found on the instructors bench when preparing your standards as the volumes are too large for the microcentrifuge tubes.
Vial |
Volume of diluent (mL) | Volume (mL) and source of BSA (vial) | Final BSA concentration (μg/mL) |
---|---|---|---|
A | 2.25 | 0.25 of stock | 200 |
B | 3.6 | 0.4 of A | 20 |
C | 2.0 | 2.0 of B | 10 |
D | 2.0 | 2.0 of C | 5 |
E | 2.0 | 2.0 of D | 2.5 |
F | 2.4 | 1.6 of E | 1 |
G | 2.0 | 2.0 of F | 0.5 |
H | 4.0 | 0 | Blank |
Part 2b: Prepare Working Reagent (WR) and measuring protein concentration
- Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 1.1 = total volume of WR (in mL).
- Prepare the calculated volume of WR by mixing the Micro BCA Reagent MA, Reagent MB, and Reagent MC such that 50% of the total volume is MA, 48% is MB, and 2% is MC.
- For example, if your calculated total volume of WR is 100 mL, then mix 50 mL of MA, 48 mL of MB, and 2 mL of MC.
- Prepare your WR in a 15 mL conical tube.
- Pipet 0.5 mL of each standard prepared in Part 4a into clearly labeled 1.5 mL microcentrifuge tubes.
- Prepare your protein sample by adding 990 μL of dH2O to your 10 μL aliquot of purified protein, for a final volume of 1 mL in clearly labeled 1.5 mL microcentrifuge tubes.
- Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein sample.
- Cap your tubes and incubate at 60°C in the water bath for 1 hour.
- Following the incubation, use the spectrophotometer to measure the protein concentrations of your standards and your purified protein sample.
- The cuvette filled only with water (H) should be used as a blank in the spectrophotometer.
- Measure the absorbance at 562 nm for each solution.
- Generate your standard curve by plotting the A562 for each BSA standard (B-H) vs. its concentration in μg/mL.
- Use the standard curve in its linear range (0.5 - 20 μg/mL), and its linear regression in Excel, to determine the protein concentration of purified FKBP12 in your sample.
Reagents
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