Add 100 μl of TYE Medium to the wells of a 96-well round-bottom microtiter plate (Nunc).
Transfer an inoculum from individual colonies of the selected phage library with a sterile toothpick (see Tip #1 and #2).
Incubate the plate on a shaking platform (300 rpm) at 30°C overnight (see Tip #3).
Add 50 μl of TYE (+) Glycerol to each well and mix by trituration. This is the master plate.
Transfer 2 μl from the master plate to another 96-well microtiter plate containing 150 μl of TYE Media per well.
Wrap the master plate in plastic wrap and store at -80°C.
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).
Add 50 μl of TYE Media containing 2 X 109 pfu/ml helper phage (M13K07, R408 or VCSM13) to each well (see Tip #5).
Place the plate at 37°C for 30 min (see Tip #6).
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.
Aspirate the supernatants and resuspend the bacterial pellets in 150 μl of TYE (-) Glucose (see Tip #7).
Incubate the plate on a shaking platform (300 rpm) at 30°C overnight (see Tip #3).
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.
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)
1. These bacteria are derived after several rounds of selection and represent the phage that have affinity for the antigen of interest.
2. Each microtiter plate should include the following controls:
a. A well of cells that does not contain phagemid
b. A well of cells containing pHEN1 without antibody insert
c. A well of cells containing phage displaying non-relevant antibody fragments (non-selected)
3. To reduce evaporation, microtiter plates should be placed in a closed box on top of damp paper towels. Do not place the box close to the fan of the incubator. Test the microtiter plates to ensure that shaking does not cross-contaminate wells.
4. This incubation will take approximately 2.5 hr.
5. The ratio of helper phage to bacterium should be 20:1.
6. Incubation at 37°C maintains pili. The presence of Glucose in the medium inhibits PIII protein production and allows pilus expression.
7. Add Kanamycin to 25 μg/ml for helper phage M13K07 or VCSM13. The omission of Glucose from the medium allows leaky expression of the PIII protein under the control of the LacZ promoter.
References for Phage Display Protocol
Based on the protocol by Andrew Bradbury of Los Alamos National Laboratory.
Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001)
