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Phage Display Rabbit Antibody Library Construction Kit

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I. LIST OF COMPONENTS

Phagemid: pCDisplay-4 E. coli TG1 host strain: K12 D(lac-pro), supE, thi, hsdD5/F’[traD36, proAB, lacIq, lacZDM15]. TOP10F′host strain: F′{lac lq Tn10 (tetR)}, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, araD139, Δ(ara -leu )7697, galU, galK, rpsL (strr), endA1, nupG M13K07 helper phage: ~1.0×1011pfu phages (~1.0×1012 pfu/mL) supplied in 100μL TBS with 15% glycerol. Kanamycin resistant. The sequencing primers:
VH: 5’-ACCTATTGCCTACGGCAGCCG-3’
VL: 5’-AAGACAGCTATCGCGATTGCAG-3’
Kit storage: In the kit, there are two bacterial strains in stab agar to be stored at 4 °C.  The helper phage should be kept at -20°C. There are two DNA primers to be stored at -20°C as well.

II. INTRODUCTION & LIBRARY OVERVIEW

Principle of phage display

In a phage display library, a variety of peptides, small antibodies [e.g. scFv and Fab] or proteins are displayed on the surface of filamentous phage [M13, fd, and f1 strains] or lambda phage as fusion proteins with one of the coat proteins of the phage virions, while the genetic materials encoding the peptides/proteins are housed within the virion. Using a binding-based process called bio-panning [Figure 1], a small number of phages that display proteins specifically binding to a target of interest can be rescued from a phage library that usually displays a repertoire of many billions of unique peptides/proteins. Finally, the peptides/proteins displayed by the selected phages can be identified by phage amplification and DNA sequencing.

Principle of phage display

III. MATERIALS AND METHODS FOR USING THE KIT

Media and solution
LB Medium: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave 20 minutes at 121°C.
LB Plates: LB medium + 15 g/L agarose.
Top agar: as for LB medium, but use 7 g/L Bacto-agar. Before use, melt and cool to 50°C.
SB (Super Broth) Medium: 10 g of MOPS, 30 g of tryptone, 20 g of yeast extract, 1 liter volume with ddH2O. Stir to dissolve, titrate to pH 7.0. Autoclave at 121°C for 20 minutes.
PBS: Per liter: 8 g NaCl, 0.2 g KCl, 1.7 g Na2HPO4, 0.163 g KH2PO4, pH to 7.4 with HCl.
Blocking buffer: 0.1M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3, Filter sterilize, store at 4°C.
Coating Buffer: 0.1M NaHCO3 (pH 8.6).
Conjugated second antibody: 

Human Immunoglobulin G (IgG) Fab'2 Rabbit anti-Human Polyclonal (HRP) Antibody: LifeSpan BioSciences
EIA/RIA strip-well 96-well plates: Corning, USA
Substrate: TMB and H2O2 from Pierce.
5*PEG/NaCl Dissolve in water: 200 g of polyethylene glycol-8000, 150 g of NaCl(2.5M). Bring volume to 1 liter; stir until dissolved. Filter through a 0.8 µm filter.
SOB Medium: To 950 mL of deionized water, add 20 g of tryptone, 5.0 g of yeast extract, 0.5 g of NaCl, 186 mg of KCl. Mix the solution until dissolved, and adjust the pH to 7.0 with NaOH. Adjust the volume to 1 liter. Sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle. When cooled, add 10 mL of sterile 1 M MgCl2.

SOC Medium: Add 1 mL of 1 M glucose (filter-sterilized) to 50 mL of SOB (final = 20 mM).


IV. APPENDICES


REFERENCES

  1. C. F. Barbas III, D. R. Burton, J. K. Scott, and G. J. Silverman, (2001) Phage Display: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  2. O'Brien P.M. Aitken R. Totowa, (2001) Antibody phage display: methods and protocols. In: Methods in Molecular Biology, Vol 178. Humana Press, NJ.
  3. Sachdev S. Sidhu, (2005) Phage Display in Biotechnology and Drug Discovery. CRC Press/Taylor & Francis Group, Boca Raton.

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