Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

Antibody Phage Expression Protocol Procedure

1. Add 100 μl of TYE Medium to the wells of a 96-well round-bottom microtiter plate (Nunc).
2. Transfer an inoculum from individual colonies of the selected phage library with a sterile toothpick (see Tip #1 and #2).
3. Incubate the plate on a shaking platform (300 rpm) at 30°C overnight (see Tip #3).
4. Add 50 μl of TYE (+) Glycerol to each well and mix by trituration. This is the master plate.
5. Transfer 2 μl from the master plate to another 96-well microtiter plate containing 150 μl of TYE Media per well.
6. Wrap the master plate in plastic wrap and store at -80°C.
7. Incubate the second plate on a shaking platform (300 rpm) at 37°C until the optical density at 600 nm wavelength (OD600) is approximately 0.5 (see Tip #4).
8. Add 50 μl of TYE Media containing 2 X 109 pfu/ml helper phage (M13K07, R408 or VCSM13) to each well (see Tip #5).
9. Place the plate at 37°C for 30 min (see Tip #6).
10. Centrifuge the plates at 2,700 rpm (1,200 X g) for 10 min with a Beckman™ GH-3.8 rotor and microtiter plate adaptors.
11. Aspirate the supernatants and resuspend the bacterial pellets in 150 μl of TYE (-) Glucose (see Tip #7).
12. Incubate the plate on a shaking platform (300 rpm) at 30°C overnight (see Tip #3).
13. Centrifuge the plates at 2,700 rpm (1,200 X g) for 10 min with a Beckman™ GH-3.8 rotor and microtiter plate adaptors.
14. Use 50 μl of the supernatants for ELISA assay.

Phage Display Antibody Expression Buffer Recipes

TYE (-) Glucose    5 g NaCl Prepare in 1 liter of ddH2O 10 g Bacto Yeast Extract (Difco) Add Ampicillin to 100 μg/ml 16 g Bacto Tryptone (Difco) Cool to 55°C Autoclave to sterilize TYE (+) Glycerol    Cool to 55°C 16 g Bacto Tryptone (Difco) 5 g NaCl Prepare in 1 liter of ddH2O 30% (v/v) Glycerol Add Ampicillin to 100 μg/ml 10 g Bacto Yeast Extract (Difco) Autoclave to sterilize Add Glucose to 2% (w/v) TYE Media    Cool to 55°C 16 g Bacto Tryptone (Difco) 5 g NaCl Prepare in 1 liter of ddH2O Add Ampicillin to 100 μg/ml 10 g Bacto Yeast Extract (Difco) For plates, add 15 g Bacto Agar (Difco) before autoclaving Autoclave to sterilize Add Glucose to 2% (w/v)

Antibody Phage Expression Method Materials Needed:

• Sodium Chloride
• Bacto Tryptone
• Glycerol
• Ampicillin
• Glucose
• Bacto Yeast Extract

Protocol Tips

References for Phage Display Protocol