Difference between revisions of "20.109(S17):Evaluation of purified protein (Day3)"

Introduction

Protocols

Part 1: Workshop with BE Communication Lab

Our communication instructors, Dr. Sean Clarke and Dr. Diana Chien, will join us today for a workshop on designing effective figures and captions.

Part 2: Visualize purified protein with polyacrylamide gel electrophoresis (PAGE)

1. 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.
2. 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.
3. 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.
4. 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.
   
5. Pry apart the plates using a spatula, and carefully transfer your gel to a staining box.
   
6. Add enough distilled water to cover the gel (~200 mL) and rinse the gel for 5 minutes on the shaker.
   
7. Repeat the rinse two more times with fresh water (~200 mL and 5-minute incubation each time).
   
8. Add 50 mL of BioSafe Coomassie, and incubate for at least 1 hour.
   
9. Empty the staining solution into the waste container in the fume hood - careful not to lose your gel!
   
10. Add 200 mL of water to your stained gel. Replace with fresh water just before leaving the lab if you have a chance.
    
11. 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 3: Measure protein concentration

Part 3a: Prepare diluted albumin (BSA) standards

1. Obtain a 0.25 mL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of diH₂O from the front bench.
2. 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.

Part 3b: Prepare Working Reagent (WR) and measuring protein concentration

1. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 1.1 = total volume of WR (in mL).
2. 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.
3. Pipet 0.5 mL of each standard prepared in Part 4a into clearly labeled 1.5 mL microcentrifuge tubes.
4. 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.
5. Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein sample.
6. Cap your tubes and incubate at 60°C in the water bath for 1 hour.
7. 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 to blank 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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