신제품 Phage Display Peptide Library Kit v2*는 affinity bead capture를 사용하는 solution-phase panning으로 기존 surface panning 보다 실험 시간이 단축되었습니다. Control panning 실험은 DYKDDDDK Mouse mAb와 Protein G Magnetic beads 사용으로 streptavidin 보다 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
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 |