[NEB] Ph.D. Phage Display Peptide Library Kit v2 (updated 2022.11)

[NEB] Ph.D. Phage Display Peptide Library Kit v2

신제품 Phage Display Peptide Library Kit v2*는 affinity bead capture를 사용한 solution-phase panning으로 기존 surface panning 보다 실험 시간이 단축되었고, DYKDDDDK Mouse mAb에 대한 panning은 신뢰할 수 있는 epitope results를 얻을 수 있습니다.

   * Ph.D.-7, Ph.D.-12 libraries는 동일하며, panning (capture) 방법이 변경되었습니다.

  • Ready to use complex phage library (~109 clones)
  • Inherent link between phenotype and genotype allows screening of billions of clones in a single microtiter well or Eppendorf tube
  • Solution-phase control panning experiment does not require blocking step, eliminating spurious plastic binders and reducing wash steps 
  • Does not require helper phage for amplification
Panning with a pentavalent peptide library displayed on pIII
Panning with a pentavalent peptide library displayed on pIII

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

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.

Ph.D.-7 Phage Display Peptide Library Kit v2

The Ph.D.-7™ Phage Display Peptide Library Kit v2 contains the Ph.D.-7 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.-7 Phage Display Peptide Library is a combinatorial library of random 7-mer peptides fused to the N-terminus of a minor coat protein (pIII) of M13 phage.

Ph.D.-C7C Phage Display Peptide Library Kit v2

The Ph.D.-C7C™ Phage Display Peptide Library Kit v2 contains the Ph.D.-C7C 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.-C7C Phage Display Peptide Library is based on a combinatorial library of random disulfide looped 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.

1. Original Kit 와 New v2 Kit 구성품 비교 표

 

Original Kit Components

New v2 Kit Components

Ph.D. Phage Display Peptide Library

E8102 or E8111 or E8121

E8102 or #E8111 or E8121

E. coli K12 ER2738

E4104

E4104

-96 gIII Sequencing Primer

S1259 (100 pmol)

S1259 (500 pmol)

-28 gIII Sequencing Primer

S1258 (100 pmol)

Streptavidin

N7023

Biotin

N7023

DYKDDDDK Mouse mAb

E8004

Protein G Magnetic Beads

S1430

2. Original Kit 와 New v2 Kit Method 비교 표

 

Original Kit Components

New v2 Kit Components

 

Surface Panning

Solution Phase Panning

Capture

Polystyrene surface (well or petri dish)

Protein G Magnetic Beads (#S1430)

Prepare Capture Surface

Overnight coating of target (Streptavidin, #N7023), 2 hr blocking

None

Binding (Selection Step)

Apply Ph.D. library to coated surface (10-60 min, RT)

Mix target (DYKDDDDK Mouse mAb, #E8004) + Ph.D. library (20 min RT). Capture phage-target complexes with Protein G Magnetic Beads (#S1430)

Wash

10 x 1ml TBST, pipet or dump

10 x 1ml TBST, pellet with magnet

Elution

1 mL of 0.1 mM Biotin (#N7024) (30 min RT)

1 mL of pH 2 Glycine Buffer (10-20 min RT), neutralize to pH 9 with TrisHCl

 

Enrich in E. coli culture (4-5 hr)

Repeat selection 1-2 more times

Sequencing and/or binding studies (-96 gIII Sequencing Primer, #S1259)

All three ready-made libraries (Ph.D.-7, Ph.D-12 and Ph.D-C7C) may contain binders to a given target. Choice of library is not dependent on target-type or downstream application. For this reason, we recommend that most customers start with either of the two linear libraries, Ph.D-7 or Ph.D-12. The exception is when a looped structure is necessary for the application, in which case the disulfide looped Ph.D.-C7C library is recommended.

The linear Ph.D-7 and Ph.D-12 libraries will have binders to most targets. For those targets that do not work with the linear libraries, the disulfide looped Ph.D-C7C library may have binders to the target of interest. The Ph.D.-7 library consists of randomized linear 7-mer peptides and may be most useful for targets requiring binding elements concentrated in a short stretch of amino acids. The Ph.D.-12 library consists of randomized linear 12-mer peptides. These 12-mer peptides have a diversity equivalent to the Ph.D.-7 library but spread over more sequence space. A structurally constrained library such as the Ph.D.-C7C library are especially useful for targets whose native ligands are in the context of a surface loop, such as antibodies with structural epitopes. Additionally, imposing structural constraints on the unbound ligand may result in a less unfavorable binding entropy, improving the overall free energy of binding compared to unconstrained ligands (O’Neil, K.T. et al 1992 Proteins 14, 509-515). A major disadvantage of structurally constrained libraries is that the constraint may “freeze out” a conformation required for target binding, preventing binding outright rather than improving affinity (McConnell, S. J. et al 1994 Gene 151, 115-118). Regardless of the library, typically only 3-5 positions are critical for binding with a target.

♦ 기존 제품 NEB #E8100S #E8110S #E8120S는 단종됩니다. (Dec 15, 2022 예정)

Discontinued products

Part No

Product Name

대체 제품

E8100S

Ph.D.™-7 Phage Display Peptide Library Kit

NEB #E8211 Ph.D.-7 Phage Display Peptide Library Kit v2

E8110S

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

NEB #E8210  Ph.D.-12 Phage Display Peptide Library Kit v2

E8120S

Ph.D.™-C7C Phage Display Peptide Library Kit

NEB #E8212  Ph.D.-C7C Phage Display Peptide Library Kit v2

* 주문 및 견적 문의는 지사/대리점으로 연락 주세요.

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