ph d 12 peptide library aliquot  (New England Biolabs)

Journal: Scientific Reports

Article Title: Identification of a peptide for folate receptor alpha by phage display and its tumor targeting activity in ovary cancer xenograft

doi: 10.1038/s41598-018-26683-z

Figure Lengend Snippet: Screening and identification of FRα binding peptides. A Ph.D.-12 phage library was used to screen FRα binding phages with four rounds of biopanning. ( A ) The enrichment of FRα binding phages were evaluated by phage recovery yields of each round selection. ( B ) Polyclonal phage ELISA using elutes after each round selection. ( C ) 94 phage clones were randomLy picked from the third round selection, and their binding affinities for FRα were analyzed individually by phage ELISA.

Article Snippet: After washing 3 times with TBS containing 0.05% (v/v) Tween 20 (TBST), 100 μL Ph.D.-12 peptide library aliquot (New England Biolabs, Inc., USA) containing 1011 phages was added to each well and the plate was incubated for 1 hour at 37 °C.

Techniques: Binding Assay, Selection, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Scientific Reports

Article Title: Discovery of a polystyrene binding peptide isolated from phage display library and its application in peptide immobilization

doi: 10.1038/s41598-017-02891-x

Figure Lengend Snippet: Polystyrene binding of PB-TUP, Ph.D.-12 peptide library, M13KE, and a non-relevant phage clone (D12) determined by ELISA. Four phage clones were added to the microtiter plates and the wells were treated with six different blocking buffers (first line) and six different washing buffers (second line), separately. ELISA values were used to evaluate the binding ability of these four phage clones to polystyrene. NFM had the best blocking effect. PBST and TBST were more effective washing buffers than the others. PB-TUP phage clone always had much higher absorbance comparing with the other phage clones. The experiments were performed in triplicates and repeated twice.

Article Snippet: 1010 pfu of possible polystyrene binding clone PB-TUP, Ph.D.-12 peptide library, M13KE (LacZa(-) wild-type M13 phage, New England BioLabs, Inc., USA), and a non-relevant phage D12 clone was added into the microtiter plates as a control group and the plates were incubated overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

doi: 10.1371/journal.pone.0058200

Figure Lengend Snippet: Selection of a peptide phage display library against W10 embryonic progenitor cells. (A) Peptide phages that bind to W10 embryonic progenitor cell line were enriched by 3 rounds of biopanning. PhD-12 phage display peptide library (2×10 11 pfu, for round 1) or amplified recovered phage (2×10 10 pfu, for rounds 2 and 3) were first adsorbed against human adult dermal fibroblasts cells and then incubated with adherent W10 cells. The phages were recovered from the cell lysate and sample phage clones were sequenced. The enriched library was amplified for further rounds of selection. (B) The percentage of input phages recovered increased with each round of selection. The percentage of input phages recovered was determined by titration of plaque forming units (pfu) in the cell lysate relative to the input pfu used for each panning round. (C) Frequency and multiple sequence alignment of peptides identified as candidate peptide phage in rounds 2 and 3 of panning generated by CLUSTAL W (2.10). (D) Phylogram based on (C) denoting peptide similarities.

Article Snippet: Selection of Cell Binding Peptides from of a Peptide Phage Display Library Peptide phage display library (Ph.D.-12, New England Biolabs) at 2×1011 pfu was adsorbed against human dermal fibroblasts (HDF) (1×106 cells grown for 48 h in gelatin-coated 10 cm-dish) for 1 h on ice in W10 growth media with 2% BSA (library volume: 5 ml).

Techniques: Selection, Amplification, Incubation, Clone Assay, Titration, Sequencing, Generated

Journal: PLoS ONE

Article Title: Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

doi: 10.1371/journal.pone.0058200

Figure Lengend Snippet: Phage binding competition with free peptide. Competition of the peptide phage with free peptide was measured using (A) Immunofluorescent detection of bound peptide phages. Chemically synthesized peptides were added to compete with binding of peptide phages to W10 progenitor cells. Cells were pre-incubated with different peptides at 100 µM or without peptide for 30 min at 4°C, followed by peptide phages (2×10 10 pfu) for an additional 1 h at 4°C. After washing, the bound peptide phages were detected by immunofluorescence. Peptide sequences are: W10-R2-11-biotin: GWVIDYDYYPMRGGGK(biotin); FITC-W10-R2-11: FITC-GWVIDYDYYPMRGGG and FITC-unrelated: FITC-NHVHRMHATPAY (B) Percentage of input phage recovered from cell lysate. Cells were pre-incubated with peptides at 5 µM or 5 nM, or without peptide for 30 min at 4°C, followed by peptide phages (2×10 10 pfu) for an additional 1h at 4°C. After washing, the recovered phage was quantified by titration. The competition is shown as percentage of no-peptide control. Values are from triplicate experiments shown as mean ± standard deviation. Competition by the corresponding free peptide was statistically significant at 5 nM and 5 µM with the exception of W10-R2-21 (only significant at 5 µM). Competition by scrambled or unrelated peptide was not statistically significant. (ANOVA with Dunnett’s multiple comparison tests; p values: *:

Article Snippet: Selection of Cell Binding Peptides from of a Peptide Phage Display Library Peptide phage display library (Ph.D.-12, New England Biolabs) at 2×1011 pfu was adsorbed against human dermal fibroblasts (HDF) (1×106 cells grown for 48 h in gelatin-coated 10 cm-dish) for 1 h on ice in W10 growth media with 2% BSA (library volume: 5 ml).

Techniques: Binding Assay, Synthesized, Incubation, Immunofluorescence, Titration, Standard Deviation

Journal: PLoS ONE

Article Title: Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

doi: 10.1371/journal.pone.0058200

Figure Lengend Snippet: Binding of peptide display phages to W10 embryonic progenitor cell line. (A) Immunofluorescent detection of bound phages. Cells were incubated with 2×10 10 phage particles for 2 h at 37°C; unbound phages were removed by washing and cells were fixed and permeabilized. Bound phages were detected by immunocytochemistry using rabbit anti-phage antibody and Alexa568-conjugated goat anti-rabbit antibody. Cell nuclei were stained using DAPI. (B) Quantitation of peptide phage cell binding. 2×10 10 pfu of each candidate or controls (RGD, Gly12 and empty phage M13KE) phages were assessed for binding on 1×10 5 W10 progenitor cells for 2 h at 37°C. Cell associated phages were recovered from cell lysates and quantified by titration. Protein in cell lysates was measured by microBCA assay. The relative binding factor (BF) is calculated as peptide phage recovery (percentage of input) relative to M13KE control phage recovery (percentage of input). Values are from triplicate experiments and shown as mean ± standard deviation. BFs for the 4 W10 peptide phage were statistical significant from the control M13KE phage (ANOVA with Dunnett’s multiple comparison tests; p values: *:

Article Snippet: Selection of Cell Binding Peptides from of a Peptide Phage Display Library Peptide phage display library (Ph.D.-12, New England Biolabs) at 2×1011 pfu was adsorbed against human dermal fibroblasts (HDF) (1×106 cells grown for 48 h in gelatin-coated 10 cm-dish) for 1 h on ice in W10 growth media with 2% BSA (library volume: 5 ml).

Techniques: Binding Assay, Incubation, Immunocytochemistry, Staining, Quantitation Assay, Titration, Standard Deviation

Journal: PLoS ONE

Article Title: Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

doi: 10.1371/journal.pone.0058200

Figure Lengend Snippet: Labeling of embryonic progenitor cell line using peptide targeted Qdot605. (A) Cell targeting by fluorescent Qdots. Qdot605-ITK-SA were complexed with an excess of chemically synthesized C-terminal biotinylated peptide; unbound peptide was removed by dialysis. W10 progenitor cells were incubated for 16 h at 37°C with 5 nM of Qdot complexes, washed and imaged using a fluorescence microscope. (B) Competition with free peptide or peptide-targeted Qdots. Cells were pre-incubated with 5nM peptide, peptide targeted Qdots, or untargeted Qdots, for 30 min at 4°C, followed by addition of peptide phage (2×10 10 pfu) for an additional 1 h at 4°C. After washing, the recovered phage was quantified by titration. The competition is shown as percentage of no-peptide control. Values are from triplicate experiments and shown as mean ± standard deviation. Competition by corresponding free peptide or peptide-Qdot complex at 5 nM was statistically significant. Competition by uncoupled Qdots was not statistically significant (ANOVA with Dunnett’s multiple comparison tests; p values: *:

Article Snippet: Selection of Cell Binding Peptides from of a Peptide Phage Display Library Peptide phage display library (Ph.D.-12, New England Biolabs) at 2×1011 pfu was adsorbed against human dermal fibroblasts (HDF) (1×106 cells grown for 48 h in gelatin-coated 10 cm-dish) for 1 h on ice in W10 growth media with 2% BSA (library volume: 5 ml).

Techniques: Labeling, Synthesized, Incubation, Fluorescence, Microscopy, Titration, Standard Deviation