Product Class: Kit

Ph.D™.-12 Phage Display Peptide Library Kit v2

This product is a direct replacement for NEB #E8110S, Ph.D.™-12 Phage Display Peptide Library Kit


Catalog #E8210

Product Introduction

The Ph.D.-12™ Phage Display Peptide Library Kit v2 contains the Ph.D.-12 Phage Display Peptide Library, a DYKDDDDK Mouse monoclonal antibody and Protein G Magnetic Beads for a panning control experiment, and enough -96gIII sequencing primer for >50 sequencing reactions. The Ph.D.-12 Phage Display Peptide Library is a combinatorial library of random 12-mer peptides fused to the N-terminus of a minor coat protein (pIII) of M13 phage. The library consists of ~109 electroporated (i.e., unique) sequences. 

  • Ready to use complex phage library (~109 clones)
  • Monoclonal antibody and Protein G Magnetic Beads included for new control panning experiment
  • Panning against DYKDDDDK Mouse monoclonal antibody yields more reliable epitope results than former target protein (streptavidin)
  • Does not require helper phage for amplification
  • Inherent link between phenotype and genotype allows screening of billions of clones in a single microtiter well or Eppendorf tube

Product Information

Description

Phage display describes a selection technique in which a library of peptide or protein variants is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside (1-3). This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called panning (4). In its simplest form, panning is carried out by incubating a library of phage-displayed peptides on a plate (or bead) coated with the target, washing away the unbound phage, and eluting the specifically bound phage (Figure 1). The eluted phage are then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences. After 3–4 rounds, individual clones are characterized by DNA sequencing and binding assays.

The Ph.D.-12 Phage Display Peptide Library Kit v2 is based on a combinatorial library of random dodecapeptides fused to the N-terminus of a minor coat protein (pIII) of M13 phage (5-9). The peptide is followed by a short spacer (Gly-Gly-Gly) which directly precedes the wild-type pIII sequence. The library consists of ~109 electroporated sequences amplified once to yield approximately 100 copies of each sequence in 10 μl of the supplied phage.

Displayed Peptide Form: X12-GGG-native M13 pIII

Figure 1: Panning with a pentavalent peptide library displayed on pIII.



 
This product is related to the following categories:
Protein Tools Products,
Phage Display Products,
This product can be used in the following applications:
Phage Display,
Protein Analysis Tools

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Features

  • Ready to use complex phage library; inherent link between phenotype and genotype allows screening of billions of clones in a single microtiter well or Eppendorf tube
  • Control panning experiment does not require blocking step and eliminates spurious plastic binders
  • Control panning experiment with Protein G Magnetic Beads reduces tedium of plate/well washing steps 
  • Length of random region (12-mer) allows for intramolecular secondary structure formation as well as sequence gaps between defined positions
  • Does not require helper phage for amplification
  • Discover peptide binders in less than one week with straight-forward molecular biology and sterile culture techniques
  • Supplied -96 gIII Sequencing Primer (500 pmol) allows for >50 sequencing reactions

Product Notes

  1. Recommended long term storage (>30 days) for E. coli K12 ER2738 is -80°C.
  2. Protein G Magnetic Beads should be stored at 4°C to prevent damage from freezing. 
  3. The original kit (NEB #E8110) and the new version 2 (v2) kit (NEB #E8210) both contain the same Ph.D.-12 Phage Display Peptide Library component (NEB #E8111).
  4. M13 are compatible with most buffer conditions; however, elution buffers may be limited by E. coli culturing conditions needed for amplifications. Amplifications for enriching phage pools may be eliminated if using next generation sequencing strategy. Scan current literature for protocols.
  5. Supplied -96 gIII Sequencing Primer (500 pmol, 5´-CCCTCATAGTTAGCGTAACG-3 ´) allows for >50 sequencing reactions.

References

  1. Sidhu, S.S. (2000). Curr. Opin. Biotechnol.. 11, 610-616.
  2. Rodi, D.J. and Makowski, L. (1999). Curr. Opin. Biotechnol. . 10, 87-93.
  3. Wilson, D.R. and Finlay, B.B. (1998). Can. J. Microbiol.. 44, 313-229.
  4. Parmley, S.F. and Smith, G.P. (1998). Gene. 73, 305-313.
  5. Scott, J.K. and Smith, G.P. (1990). Science. 249, 386-390.
  6. Whaley, S.R. et al. (2000). Nature. 405, 665-668.
  7. Noren, K.A. and Noren, C.J. (2001). Methods. 23, 169-178.
  8. Rozinov, M.N. and Nolan, G.P (1998). Chem. Biol.. 5, 713-728.
  9. Rodi, D.J. et al. (1999). J. Mol. Biol.. 285, 197-203.

Protocols, Manuals & Usage

Protocols

  1. Quick Start Protocol PhD Phage Display Peptide Library Kit v2
  2. Panning Protocol 1: Solution-phase Panning with Affinity Bead Capture
  3. Panning Protocol 2: Surface-Phase Panning (Direct Target Coating)
  4. Phage ELISA Binding Assay with Direct Target Coating
  5. Phage Titering
  6. Plaque Amplification for ELISA Samples
  7. Post Panning Protocol 1: Rapid Purification of Single-Stranded DNA Templates for Sequencing Reactions

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

FAQs & Troubleshooting

FAQs

  1. What is the difference between the original and version 2 (v2) Phage Display kits?
  2. Which of the three ready-made libraries should I choose?
  3. What is the difference between the three ready-made libraries?
  4. Can a different bacterial strain be used with the Ph.D.™ Phage Display?
  5. No plaques are visible when titering using a Ph.D.™ Phage Display Library.
  6. I am using Ph.D.™ Phage display and the amplified phage titer is low.
  7. I am using Ph.D.™ Phage Display and the phage DNA templates do not yield a readable sequence.
  8. I am using Ph.D.™ Phage Display and the sequencing templates do not run where they should on a gel.
  9. I am using Ph.D.™ Phage Display and after 4 or more rounds of panning all clones are wild-type phage (white plaques).
  10. When performing an experiment using Ph.D.™ Phage Display, the ELISA indicates that background binding to the plate is as high as binding to the target.
  11. When using the Ph.D.™ Phage Display, panning yielded a consensus sequence, but no ELISA signal.
  12. Where can I find references for Ph.D.™ phage display libraries?
  13. What is the pIII sequence for an insertless clone? What is at the N-terminus of a PhD clone?
  14. How are the PhD Phage Display Libraries cloned?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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