New England Biolabs, E8211S, 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. The library consists of ~10 Related Categories Protein Tools,, Phage Display Applications Phage Display,, Protein Analysis Tools FAQ Q: What is the difference between the original and version 2 (v2) Phage Display kits? A: The exact differences between the original version and the v2 kits are outlined in the table below. The Ph.D.-7, Ph.D.-12 and Ph.D.-C7C libraries have not changed, and their product numbers remain the same. The new v2 kits include a streamlined and modernized solution-phase panning control experiment that requires fewer experimental steps, whereas the original kits recommend a surface panning experiment. Additionally, the -96 gIII Sequencing Primer is now supplied at a 10-fold higher concentration (10 pmol/ml) to better align with commercial sequencing facility submission guidelines. The -28 gIII Sequencing Primer has been removed because the annealing location is too close to the Ph.D. random region to be useful in modern sequencing technologies. Original Kit Components Name Product # Amount Concentration Ph.D. Phage Display Peptide Library E8102 or E8111 or E8121 1 x 0.1 ml 1 x 1013 pfu/ml E. coli K12 ER2738 E4104 1 x 0.2 ml -96 gIII Sequencing Primer S1259 100 pmol 1 pmol/ml -28 gIII Sequencing Primer S1258 100 pmol 1 pmol/ml Streptavidin N7023 1 x 1.5 mg Biotin N7024 1 x 0.1 ml 10 mM New v2 Kit Components Name Product # Amount Concentration Ph.D. Phage Display Peptide Library E8102 or E8111or E8121 1 x 0.1 ml 1 x 1013 pfu/ml E. coliK12 ER2738 E4104 1 x 0.2 ml -96 gIII Sequencing Primer S1259 500 pmol 10 pmol/ml DYKDDDDK Mouse mAb E8004 1 x 0.015 ml Protein G Magnetic Beads S1430 1 x 0.15 ml Original Kit Method New v2 Kit Method 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) Q: Which of the three ready-made libraries should I choose? A: 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. Q: What is the difference between the three ready-made libraries? A: 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. Q: Can a different bacterial strain be used with the Ph.D.™ Phage Display? A: In theory other F+ strains containing the supE suppressor mutation (such as XL1-Blue and DH5αF´) should work with our phage display system. However, we have not tested these strains with our libraries and do not know whether here will be any subtle effects on the expression or transport of certain peptides out of the cell. Since the Ph.D. libraries are made in ER2738 (NEB #E4104S), we know that all the peptides in the libraries can be successfully expressed in this strain. Therefore, we recommend this strain over any other one. Q: No plaques are visible when titering using a Ph.D.™ Phage Display Library. A: Unlike lambda, M13 is a non-lytic phage and does not produce clear plaques. M13 plaques are areas of diminished cell growth, not lysis, and consequently can be difficult to see. Try holding the plate up to a light. Also, since the vector used to prepare the library carries the lacZα gene, plaques will be blue, and easier to see, when using an alpha complementing strain such as the supplied E. coli K12 ER2738 and plating on Xgal/IPTG plates. Also, be sure the dilution range is appropriate for the phage you are titering. For amplified phage, plate 10 µL of 1:109 - 1:1011 dilutions; for unamplified panning eluates, try 1:10 - 1:104 dilutions for early rounds, 1:104-1:107 for later rounds. If the phage is not sufficiently dilute, the plaques will be confluent on the plate and it will look like there are no plaques at all (or a bluish tinge when using Xgal plates). Occasionally after PEG precipitation, the phage will clump and not dilute properly. As a result, you might have a plate containing too many plaques merged together. Make sure to give the phage ample time to resuspend after precipitation (> 1 hour) and vortex each dilution tube very well (~10 seconds). Q: I am using Ph.D.™ Phage display and the amplified phage titer is low. A: In order for M13 phage to be efficiently amplified, it is critical that cultures be well aerated, and that cultures be infected early in their growth phase. We recommend amplification in 20 mL cultures in 250 mL Erlenmeyer flasks, in a shaker set to 250 rpm. Amplification in smaller vessels, such as 50 mL conical tubes, will result in much lower yields of amplified phage. M13 phage should either be added to an early-log culture, A600 < 0.01, or to a 1:100 dilution of an overnight culture. Yield of amplified phage is maximal after 4.5-5 hours at 37°C; longer incubation may result in deletions and is not recommended. If carrying out nonspecific elution with pH 2.2 glycine buffer, the eluted phage must be neutralized as described in the Manual prior to amplification. Q: I am using Ph.D.™ Phage Display and the phage DNA templates do not yield a readable sequence. A: The Rapid Purification of Single-Stranded DNA Templates for Sequencing Reactions protocol should provide single-stranded template of sufficient purity for sequencing reactions. The procedure should be followed exactly as described in the manual: prolonged ethanol precipitation, precipitation at 20° C or centrifugation longer than 10 minutes will result in co-precipitation of salt and phage proteins, which will inhibit sequencing. Additionally, it is crucial that the phage pellet is thoroughly suspended in the iodide buffer prior to adding ethanol. If problems persist, or if another sequencing method is used, a phenol:chloroform extraction step can be added: Following suspension in Iodide Buffer, add 2 volumes of TE, extract once with phenol:chloroform (1:1) and once with chloroform, and ethanol precipitate. 5 µL of suspended template (approximately 0.5 µg) should be sufficient for sequencing; quantitation should be confirmed by agarose gel electrophoresis using 0.5 µg single stranded M13 DNA (NEB #N4040S) as a standard. Q: I am using Ph.D.™ Phage Display and the sequencing templates do not run where they should on a gel. A: The sequencing templates prepared by the method in the manual are single-stranded (approx. 7250 nucleotides), and as a result will not line up with double-stranded markers of the same length. The apparent size will vary depending on the applied voltage, ethidium and agarose concentration in the gel, and whether TBE or TAE is used as running buffer. We strongly recommend using single-stranded M13 DNA (e.g. single-stranded M13mp18, NEB #N4040) as a marker. Q: I am using Ph.D.™ Phage Display and after 4 or more rounds of panning all clones are wild-type phage (white plaques). A: In a typical round of biopanning, ~2 x 1011 input phage are reacted with the target, and between 103 and 107 total phage are eluted off following washing. This corresponds to an enrichment of 104 to 108-fold per round. Since the library contains ~2 x 109 different clones, the eluted pool of phage should in theory be fully enriched in favor of binding sequences after only 2 or 3 rounds. Once this point is reached, further rounds of amplification and panning will result only in selection of phage that have a growth advantage over the library phage. For example, vanishingly small levels of contaminating environmental wild-type phage (less than one part per billion) will completely overtake the pool if too many rounds of amplification are carried out, regardless of the strength of the in vitro selection. Q: 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. A: If panning against a polystyrene plate coated with the target (Surface-Phase Panning or Direct Target Coating), it is possible to inadvertently select peptides that specifically bind the polystyrene surface (see Adey, N. B. et al. (1995) Gene 156, 27-31). These peptides will yield identical ELISA signals in the presence and absence of target since the ELISA plate is also made of polystyrene. Such "plastic binders" are typically rich in aromatic residues (Phe, Tyr, Trp, His), which often alternate (the sequence FHWTWYW is a plastic binder discovered and characterized at NEB). Selection of plastic binders often occurs in the absence of a strong target preference for peptide sequences present in the library: other libraries may yield the desired target specific sequences. Selection of polystyrene-specific peptides can be avoided by using the Solution Phase Panning method when possible, for a given target. The phage is reacted with the target in solution, and the phage-target complexes are then captured onto beads that specifically bind the target. Q: When using the Ph.D.™ Phage Display, panning yielded a consensus sequence, but no ELISA signal. A: When characterizing phage clones by the ELISA protocol in the manual, it is difficult to add more than 1012 virions per 100 µL well. This corresponds to a phage concentration of only 16 nM. At this concentration, an unambiguously positive ELISA signal can only be observed if the binding affinity is in the micromolar range or better. The iterative nature of phage selection permits identification of ligands with a broad range of affinities, from subnanomolar to 1 millimolar, so lower affinity ligands will not show a positive ELISA signal. In this case it is necessary to increase the concentration of the selected ligand, either by synthesing a peptide corresponding to the selected sequence (be sure to include the spacer sequence GGGS at the C-terminus, and amidate the C-terminal carboxylate if possible), or by expressing the selected sequence as an N-terminal fusion to a smaller protein. Alternatively, a sandwich ELISA can be carried out in which the selected phage is immobilized and an excess of target applied in the liquid phase. This procedure requires an antibody against the target protein, or some other means of detecting bound target protein. Coat the wells overnight with anti-M13 antibody (no HRP), wash, and add serial dilutions of each phage clone (one clone per row). After 1 hour, wash away unbound phage and add an excess of target protein (0.1 - 1 µM) in TBST. Incubate 1-2 hours at RT, wash away unbound target, and detect bound target with an enzyme-linked antibody. Q: Where can I find references for Ph.D.™ phage display libraries? A: New publications are being published all the time. Search the literature with ‘peptide phage display’ with or without your application of choice and you will find references for our libraries. Also, check out references for specific Applications and as well as seminal Publications links here. Also, we have a searchable database of more recent references accessible from the bottom right of our homepage through Publications. Q: What is the pIII sequence for an insertless clone? What is at the N-terminus of a PhD clone? A: The open reading frame for native gIII sequence begins with 5’-GTG AAA AAA TTA … (see GTG start in review Kozak, M. (1999) Gene 234, 187-208 Initiation of translation in prokaryotes and eukaryotes; van Wezenbeek, P.M.G.F. et al (1980) Gene 129-148). The N-terminus of pIII includes a leader sequence necessary for processing the newly translated protein in the cell. The mature peptide as displayed on M13 phage has been cleaved at the leader peptidase site. Note, the random region will be in the same reading frame as the leader sequence. gIII translation for M13KE or an insertless clone: MKKLLFAIPLVVPFYSHS // AETVES gIII translation for a PhD clone (X is a randomized position): Ph.D.-12 clone #E8210S, E8111L MKKLLFAIPLVVPFYSHS // X12-GGGS-AETVES…pIII→ Ph.D.-7 clone #E8211S MKKLLFAIPLVVPFYSHS // X7-GGGS-AETVES… pIII→ Ph.D.-C7C clone #E8212S MKKLLFAIPLVVPFYSHS // ACX7C-GGGS-AETVES…pIII→ An additional note, a Ph.D. clone will have 5 copies of the same pIII protein, i.e. (Ph.D.-7) X7-GGGS-AETVES…, at one end of the cylindrical particle. // indicates leader peptidase cleavage site Q: How are the PhD Phage Display Libraries cloned? A: The figure below shows the cloning scheme. Please refer to our PhD Cloning System NEB #E8101S manual for full details. See also Noren, C.J. and Nore, K.A. 2001 Methods 23, 169-178. doi:10.1006/meth.2000.1118 Q: Where can I find the entire genome sequence for the parent vector, M13KE, used to clone Ph.D. Peptide Display Libraries? A: The DNA Sequences and Maps Tool page has the nucleotide sequence in several formats as well as a map of M13KE. Note, NEB #E8101S contains double-stranded, also known as replicative form (RF), M13KE DNA (20 ug).