Protein Protocols Protein Protocols

1064 Ehrlich, Berthold, and Bailon
5. Separate biotin from biotinylated protein with PBS.
6. Collect 1-mL fractions.
7. Measure protein absorbance at 280 nm or by protein assay.
8. Pool fractions containing protein.
9. Store biotinylated protein at 4°C until ready for use.
10. Use the HABA method to determine amount of biotinylation (see kit instructions).
3.1.2. Biopanning Against Purified Immobilized Target (see Note 7)
3.1.2.1. TARGET IMMOBILIZATION
The following method can be used to immobilize target proteins by hydrophobic
adsorption on polystyrene plates whereby the target is coated onto the plate and then
excess target is removed.
1. Add 1.5 mL (150 µL of 100 µg target/mL) of immobilization buffer to each 65 × 15-mm
Petri dish (96-well microtiter plate).
2. Swirl plate repeatedly to ensure that plate is completely coated with target solution.
3. Incubate plate(s) overnight with gentle shaking in a container containing wet paper
towels at 4°C.
4. Carefully remove target solution by aspiration through a sterile needle.
5. Fill each plate (well) completely with a sterile solution of blocking buffer containing
5 mg/mL of BSA or target (if available) and 0.02% NaN3. 6. Incubate for 1 h at 4°C.
7. Remove solution by aspiration as described in step 4.
8. Wash plates (wells) by rinsing with biopan buffer.
9. Repeat wash step 5×.
3.1.2.2. AFFINITY SELECTION
1. Add 1–2 × 1011 phage to 1 mL (100 µL) of biopan buffer/plate (well).
2. Pipet phage solution onto plates (wells).
3. Incubate for 60 min at room temperature with gentle rocking.
4. Carefully remove phage solution from plates (wells).
5. Wash plates (wells) 10× with biopan buffer.
6. Elute bound phage specifically (or nonspecifically) by adding 1 mL (100 µL) of target
ligand (0.1–1 mM) or 100 µg of free target in biopan buffer (elution buffer) to plates (wells).
7. Incubate with gentle rocking for 60 min (10 min) at room temperature.
8. Collect eluates in microcentrifuge tubes.
9. Neutralize nonspecific eluate with 150 µL (15 µL) of 1 M Tris-HCl, pH 9.1.
10. Titer 1 µL of eluate (see Subheading 3.3.1.).
11. Amplify rest of eluate or store at 4°C (see Subheading 3.3.2.).
12. Repeat additional rounds of biopanning using the same pfu (1–2 × 1011 ) as described
in step 1.
13. Pick plaques from the last round (unamplified) phage titer for amplification and isolation
of single-stranded bacteriophage M13 DNA (see Subheading 3.3.3.).
3.1.3. Biopanning Against Biotinylated Target
The described protocol is a modification of that described by Smith and Scott (27).
3.1.3.1. IMMOBILIZATION OF STREPTAVIDIN
Immobilize Streptavidin as described in Subheading 3.1.2.1. Add 0.1 µg/mL of
Streptavidin to the blocking buffer to complex any biotin that may be contained in
the BSA.