20.109(S17):Evaluation of purified protein (Day3)

20.109(S17): Laboratory Fundamentals of Biological Engineering

Schedule Spring 2017 Announcements Assignments Homework Communication
1. High-throughput ligand screening 2. Gene expression engineering 3. Biomaterials engineering

Introduction

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

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.

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