Immobilized Antigen Phage Antibody Selection Protocol

Immobilized Antigen Phage Antibody Selection Protocol

Detailed Protocol for the selection of Phage Antibodies using Immobilized Antigen

1. Streak out bacteria from a frozen stock of DH5αF' or TG1 cells with an inoculation loop onto a plate containing solid Minimal Media.
2. Incubate the plate overnight at 37°C.
3. Isolate a single colony with an inoculation loop and inoculate 10 ml of LB in a sterile culture tube.
4. Incubate the culture on a shaking platform (300 rpm) at 37°C until the optical density at 600 nm wavelength (OD600) is approximately 0.5 (6 to 8 hr).
5. While the bacteria are growing, incubate a sterile Nunc MaxiSorp™ Immuno™ tube with 2 ml of Antigen Solution overnight at room temperature (see Tips #1).
6. Remove the Antigen Solution and wash the tube with 4 ml of PBS (see Tips #2):  Pipette 4 ml of PBS to the inner wall of the tube, Aspirate the solution.  
7. Repeat Step #6 two more times.
8. Add 4 ml of MPBS to the tube and incubate at 37°C for 1 hr.
9. Repeat Step #6 three times.
10.  Add 1 ml of 4% MPBS containing phage antibodies diluted to 1012 to 1013 infectious particles from the preparation protocol  (see Tips #3).
11.  Cap the tube or cover with parafilm, and incubate with end-over-end mixing on a rotary wheel at room temperature for 30 min.
12. Place the tube upright in a tube rack and incubate for 1.5 hr.
13. Remove the solution of phage antibodies and wash the tube with PBST as in Step #6.
14. Repeat Step #6 two times.
15.  Add 1 ml of 100 mM Triethylamine.
16. Cap the tube or cover with parafilm, place it in a tube rack, and incubate on a rotary platform for 8 min (see Tips #4).
17. Transfer the solution to a new plastic tube (not Immuno™ tube) and immediately add 0.5 ml of 1 M Tris-HCl, pH 7.4. Mix to neutralize the Triethylamine.
18. Add 0.75 ml of the neutralized solution to the 10 ml culture of bacteria from Step #4 and mix briefly.
19. Stand the culture at 37°C for 30 min.
20. Transfer 1 μl to a plate containing solid TYE Media (see Tips #5).
21. Pellet the bacteria by centrifuging the culture in a Sorvall™ SS-34 rotor at 5,000 rpm (2,700 X g) for 10 min.
22. Resuspend the pellet in 0.5 ml of LB.
23. Spread 250 μl portions onto two 150 mm plates containing solid TYE Media.
24. Incubate the large plates overnight at 37°C.
25. Pipette several ml (10 ml or more) of TYE Media (+) Glycerol onto each plate and triturate to collect all of the colonies into a centrifuge tube.
26. Inoculate a 10 ml culture TYE Media with a portion of the collected colonies so that the OD600 is *less equal*0.05.
27. Prepare bacteriophage with helper phage as described , with the culture volumes scaled down accordingly (see Tips #6).
28. Following Protocol ID#2204, resuspend the phage prep in a final volume of 1 ml of PBS.
29. Titer the phage with TG1 bacteria as described.
30. Repeat Steps #1 through #29 a total of two to four times. In each successive round of selection using this protocol, increase the stringency of the wash steps by washing twenty times with PBST and twenty times with PBS (Steps #13 and #14) before phage elution in Step #15 (see Tips #7).

Buffers for Phage Display

Minimal Media		1 g Ammonium Sulfate ((NH4)2SO4) 10 ml of 20% (w/v) Dextrose 0.5 g Sodium Citrate, Dihydrate 15 g Bacto Agar (Difco) Add 1 ml of 1 M Magnesium Sulfate 0.5 ml of 1% (w/v) Vitamin B1 4.5 g Potassium Phosphate, Monobasic (KH2PO4) Pour into petri dishes Prepare in 1 liter of ddH2O Autoclave the solution and cool to 50°C 10.5 g Potassium Phosphate, Dibasic (K2HPO4)
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 M Tris-HCl, pH 7.4
100 mM Triethylamine
PBST		0.1% (v/v) Tween 20 Prepared in PBS
4% MPBS		4% (w/v) Non-Fat Milk Powder (Carnation™) Prepared in PBS
2% MPBS		Prepared in PBS 2% (w/v) Non-Fat Milk Powder (Carnation™)
Antigen Solution		10 μg/ml to 1 mg/ml Antigen in PBS
PBS		See Protocol ID#2152 for preparation 4.3 mM Sodium Phosphate, Dibasic (Na2HPO4) 137 mM Sodium Chloride (NaCl) 2.7 mM Potassium Chloride (KCl) 1.4 mM Potassium Phosphate, Monobasic (KH2PO4)
LB		5 g NaCl Prepare in 1 liter of ddH2O 10 g Bacto Tryptone (Difco) Autoclave to sterilize 5 g Bacto Yeast Extract (Difco)
TYE Media (+) Glycerol		TYE Media supplemented with 10% (v/v) Glycerol
20% (w/v) Dextrose		Filter to sterilize 20% (w/v) D-Glucose
1 % (w/v) Vitamin B1		Filter to sterilize and store at 4°C 1% (w/v) Thiamine-HCl
1 M Magnesium Sulfate		1 M Magnesium Sulfate (MgSO4)

 Materials Needed for Phage Display Selection:

Tips for Phage Display Antibody Selection:

References for Phage Display Selection: