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)

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.

1. 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.
2. Transfer 15 μL from each of the wash flow-through samples into a labeled 1.5 mL eppendorf tubes.
3. Transfer 15 μL of your purified, dialyzed protein solution into a labeled 1.5 mL eppendorft tube.
4. Add 3 μL of Laemmli sample buffer to each of the aliquots (cell lysates, wash flow-throughs, and purified protein).
5. 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.
6. 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.
7. Record the order in which you will load your samples and molecular weight standards in the polyacrylamide gel.
8. 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.
9. Your samples will be electrophoresed at 200 V for 30-45 min.
10. Following electrophoresis, use the spatula to carefully pry apart the plates that encase your polyacrylamide gel.
11. Transfer your polyacrylamide gel to a staining box and add enough dH₂O to cover the gel.
12. Wash the gel for 5 min at room temperature on the rotating table.
13. Empty the water from the staining box in the sink.
    • Be careful that the gel does not fall into the sink!
14. Repeat Steps #12-13 a total of 3 times.
15. Add 50 mL of BioSafe Coomassie to the staining box and incubate for 60 min at room temperature on the rotating table.
16. 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!
17. Add 200 mL of dH₂O to the staining box.
18. 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

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.

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

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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