Using Phage Display to Select Proteins with High Affinity to a Target Protein
成績單
Thaw the phage library on ice. Then, dilute it to 100 times the library diversity in PBS. Add the polyethylene glycol/sodium chloride solution at 1/5th of the diluted library volume, and incubate the solution on ice for 30 minutes. Next, centrifuge the solution at 11,000 times g for 30 minutes at four degrees Celsius. Discard the supernatant, and centrifuge it for another two minutes to pull down the remaining supernatant and concentrate the phage pellet.
Gently re-suspend the phage pellet in one milliliter of PBT buffer per target protein to be analyzed, typically, four in total. Add 100 microliters of phage library to each coated well in the control plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.
Discard the PB buffer from the target plate into the sink, and dry the plate on paper towels. Transfer all 100 microliters of the phage library in the control plate to each coated well of the target plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.
Next, remove the phage library from the plate, and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops of buffer. Add 100 microliters of 0.1 molar hydrochloric acid to each coated well to elute the phage. Incubate the plate at room temperature for 5 minutes with 300 RPM orbital shaking.
After that, neutralize the pH by adding 12.5 microliters of pH 11 one molar Tris hydrochloride to each coated well. Transfer the eluted phage from all eight wells into a single 1.5-milliliter microcentrifuge tube. Pipette up and down during the transfer to make solutions homogenous, and aspirate all liquid from the wells. Add 10% BSA to the eluted phage to make the final concentration 1%.
Store the microcentrifuge tube at four degrees Celsius. This is the round one output. Prepare a seed culture for culturing the phage input for the next round of selection by inoculating five milliliters of 2YT/Tetracycline seed culture with an isolated E. coli colony from an agar plate.
Incubate it overnight at 37 degrees Celsius with 200 RPM orbital shaking. Dilute the target protein in the rounds two and three tube with an appropriate amount of PBS. Take half of the contents of rounds two and three tube and coat four wells per target protein in a 96-well binding plate for the next round.
Next, use half of the round one output to inoculate three milliliters of mid-log phase cells. Incubate it at 37 degrees Celsius for 30 minutes with 200 RPM orbital shaking. Add M13KO7 Helper Phage to a final concentration of 10 billion pfu per milliliter. Incubate it again at 37 degrees Celsius for an hour with 200 RPM orbital shaking.
After an hour, transfer the entire three milliliters of culture to 30 milliliters of 2YT/Carbenicillin/Kanamycin solution. Grow overnight at 37 degrees Celsius with 200 RPM orbital shaking.
