20.109(S17):Evaluation of purified protein (Day3)
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Contents
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)
During the previous laboratory session, you reserved an aliquot of your IPTG-induced and the uninduced cell lysate. In addition, the flow-through from the wash steps was stored. Today you will use SDS-PAGE to visualize the effectiveness of IPTG induction and the purification procedure.
- Retrieve the 15 μL aliquots of your IPTG-induced and the uninduced cell lysates you prepared during the previous laboratory session. In addition, collect the flow-through from your wash steps and your purified, dialyzed protein solution.
- Transfer 15 μL from each of the wash flow-through samples into a labeled 1.5 mL eppendorf tubes.
- Transfer 15 μL of your purified, dialyzed protein solution into a labeled 1.5 mL eppendorft tube.
- Add 3 μL of Laemmli sample buffer to each of the aliquots (cell lysates, wash flow-throughs, and purified protein).
- Boil all samples for 5 min in the water bath located in the chemical fume hood.
- Secure the caps with the cap-locks located in the fume hood to ensure that the eppendorf caps do not pop open during the boiling step as this will result in your sample escaping the tube.
- You will load all 6 samples and two molecular weight standards.
- A pre-stained ladder will be used to track the migration of your samples through the polyacrylamide gel.
- An unstained ladder with bands of known amounts of protein will be used to estimate protein concentration in your samples.
- Record the order in which you will load your samples and molecular weight standards in the polyacrylamide gel.
- When you are ready to load your samples, alert the teaching faculty.
- Please watch the demonstration closely to ensure your samples are correctly loaded and the polyacrylamide gel is not damaged during loading.
- Your samples will be electrophoresed at 200 V for 30-45 min.
- Following electrophoresis, use the spatula to carefully pry apart the plates that encase your polyacrylamide gel.
- Transfer your polyacrylamide gel to a staining box and add enough dH2O to cover the gel.
- Wash the gel for 5 min at room temperature on the rotating table.
- Empty the water from the staining box in the sink.
- Be careful that the gel does not fall into the sink!
- Repeat Steps #12-13 a total of 3 times.
- Add 50 mL of BioSafe Coomassie to the staining box and incubate for 60 min at room temperature on the rotating table.
- Empty the BioSafe Coomassie into the appropriate waste container in the chemical fume hood.
- Be careful that the gel does not fall into the waste container!
- Add 200 mL of dH2O to the staining box.
- Wash the gel for the remainder of the class on the rotating table.
- Replace the dH2O before you leave.
Tomorrow the teaching faculty will transfer your gel to fresh dH2O and take a photograph. The image will be posted to the Discussion tab of the Mod 1 overview page.
Part 3: Measure protein concentration
Part 3a: 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 3b: 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 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
Next day:
Previous day: Purification of induced protein