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Phage Display of Peptides in Ligand Selection 115
6. Blotto: Skim milk powder (any commercial brand) diluted in PBS.
7. Super broth (SB) media: 30 g Tryptone, 20 g Yeast extract, 10 g 3-(N-
Morpholino)-propanesulfonic acid (MOPS), to 1 l with dH 2 O and autoclave.
8. Yeast tryptone (YT) media: 16 g Tryptone, 10 g Yeast extract, 5 g NaCl, to 1 l
with dH 2 O and autoclave.
9. YT plates: the same as in step 7 with the addition of 15 g bacto-agar and
tetracycline (see step 10) when cooled to approximately 50ºC. Store plates in the
dark as tetracycline is light sensitive.
10. Tetracycline: 40 μg/ml final concentration for plates and liquid media.
11. Minimal media plates: 15 g bacto-agar in 750 ml dH 2 O, autoclave and when
cooled add 200 ml of 5 × M9 salts (5 × M9 salts: 16.9 g Na 2 HPO 4 , 7.5 g
KH 2 PO 4 , 1.25 g NaCl, 2.5 g NH 4 Cl, 500 ml dH 2 O, autoclave), 20 ml of 20%
glucose (filter sterilized), 0.5 ml of 1% thiamine-hydrochloride (filter sterilized)
and 1 ml of 20% MgCl 2 .
12. K91 Escherichia coli cells starved culture on minimal media, culture a fresh K91
plate every week.
13. Maxisorp microtiter plates (Nunc), these are recommended for high levels of
protein binding.
14. Centrifuge tubes (250 ml clear polypropylene) autoclaved.
15. One-liter flasks autoclaved, baffled flasks are recommended.
2.3. Preparation of a Peptide Affinity Column
1. N-hydroxysuccinimide (NHS)-activated Sepharose 4 fast flow media (Pharmacia
Biotech). This resin has been specially developed for coupling of peptides to a
solid matrix. It has a highly stable 6-aminohexanoic acid spacer arm which can
form an amide linkage with the primary amino group of peptides.
2. Coupling buffer: 0.1 M NaHCO 3 , 0.5 M NaCl, pH 7.5
3. In order to maintain the maximum binding capacity of the resin, all solutions
should be pre-chilled (0–4ºC) and prepared prior to coupling the ligand.
2.4. Affinity Chromatogaphy Using a Peptide Column
1. Wash buffer: 0.1 M boric acid, 0.5 M NaCl, 0.05% Tween 20, pH 8.5.
2. Elution buffer: 0.1 M glycine, pH 2.2.
3. Methods
3.1. Panning a Random Peptide Library for a Peptide Mimotope
(See Note 1)
1. Coat 10 wells of an ELISA plate (Nunc Maxisorp) with 100 μl antibody at 5–10
g/ml diluted in coating buffer overnight at 4ºC.
2. Inoculate 10 ml YT media with a colony of K91 cells and grow until log phase
(∼OD = 0.6 at 600 nm) at 37ºC shaking vigorously.