Purified MAbs 1A7 and 4E2 were selected for epitope mapping under three rounds of biopanning with the Ph.D-12 TM Phage Display Peptide Library Kit (New England BioLabs, Cambridge, MA, USA), as previously described [33 (link)]. Briefly, 96-well plates were coated with purified MAbs and incubated with blocking buffer. The phage library was then added to the plate and incubated for 1 h. After five washes with TBS buffer, 1 M Tris-HCl was added to the plate to elute the bound phages. The phages were then amplified and titred on LB/IPTG/Xgal plates for selection. The ratio of output to input was calculated as the titre of the amplified output phages to the titer of the input phages.
Ph d 12tm phage display peptide library kit
Manufactured by New England Biolabs
Sourced in United States
The Ph.D-12TM Phage Display Peptide Library Kit is a laboratory product designed for screening and identifying peptides that bind to target molecules. The kit provides a collection of randomized 12-amino acid peptide sequences displayed on the surface of M13 bacteriophage particles.
Automatically generated - may contain errors
Lab products found in correlation
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Bacto yeast extract, by Merck Group (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii dye, by Merck Group (1 mentions)
Bacto tryptone, by Merck Group (1 mentions)
M13ke control phage, by New England Biolabs (1 mentions)
7 ethyl 10 hydroxycamptothecin sn38, by Biosynth (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
7 protocols using ph d 12tm phage display peptide library kit
1
Epitope Mapping of Monoclonal Antibodies
2
Epitope Mapping of Monoclonal Antibody 3B7
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Isotypes IgG mAb identified by Mouse Immunoglobulin isotyping kit was used for epitope mapping. mAb 3B7 was first purified from mouse ascites fluid using protein G agarose (Invitrogen), according to manufacturer instructions. The concentration of purified IgG was determined by measuring absorbance at 278 nm. The epitope was mapped with purified mAb 3B7 by using the Ph.D-12TM Phage Display Peptide Library Kit (New England BioLabs), as previously described12 (link), 28 (link), 29 (link). Three rounds of biopanning were performed to select phage clones. Briefly, each well of a 96-well plate was coated with 10 μ g/mL of purified 3B7 and incubated with blocking buffer. The phage library was then added to the plate and incubated for 1 hour. After five washes with TBS buffer, 1 M Tris-HCl was added to the plate to elute the bound phages. The phages were then amplified and titred on LB/IPTG/Xgal plates for selection.
3
Epitope Mapping Using Phage Display
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The epitope was mapped with purified 3B6 mAb using the Ph.D-12TM Phage Display Peptide Library Kit (New England BioLabs), as previously described24 (link)25 (link). Three rounds of biopanning were performed. Briefly, each well of a 96-well plate was coated with 10 μg/mL of purified 3B6 mAb and incubated with blocking buffer. The phage library was then added to the plate and incubated for 1 hour. After five washes with TBS buffer, 1 M Tris-HCl was added to the plate to elute the bound phages24 (link)25 (link). The phages were then amplified and titred on LB/IPTG/Xgal plates for selection. The ratio of output to input was calculated as the titre of the amplified output phages to the titre of the input phages.
4
Phage Display Peptide Library Protocol
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The Ph.D. -12TM phage display peptide library kit containing E. coli host strain ER2738 and M13KE control phage (New England BioLabs, Ipswich, MA) was used. The phage display library contained random peptides constructed at the N-terminus of the coat protein (pIII) of M13 phage. The titer of the library was 0.5 to 2 × 1013 plaque-forming unit. Horseradish peroxidase/anti-M13 monoclonal conjugate antibody (Abcam, Cambridge, UK) was used. Fetal bovine serum and trypsin were obtained from Thermo Fisher Scientific (Waltham, MA). The DNA sequencing primer was synthesized at Cosmogenetech (Seoul, Korea). Bacto-tryptone, bacto-yeast extract, and NaCl were obtained from Sigma-Aldrich (St. Louis, MO). 1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Echelon Biosciences (Salt Lake City, UT). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)(DSPE-mPEG) (molecular weight, 3000) was purchased from Nanosoft Polymers (Winston-Salem, NC). 7-Ethyl-10-hydroxycamptothecin (SN38) was purchased from Carbosynth (San Diego, CA). Hematoporphyrin (HPP), soybean oil, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbo cyanine perchlorate (DiI dye) were purchased from Sigma-Aldrich (St. Louis, MO).
5
Isolation and Identification of V. mimicus
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The V. mimicus 04–14 isolate was obtained from Ctenopharyngodon idella with ascites disease and then identified using the API 20 NE system and 16S rRNA gene sequencing as in our earlier studies [6 ]. The isolate was cultured in brain heart infusion broth (BHI; Beijing Solarbio Science & Technology Co., Ltd., China) at 30°C. The Ph.D.-12TM Phage Display Peptide Library Kit containing E. coli host strain ER2738 and _96 gIII sequencing primer was purchased from New England BioLabs.
6
Monoclonal Antibody Epitope Mapping
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The epitope was mapped by using the Ph.D-12TM Phage Display Peptide Library Kit (New England BioLabs Inc.) and mAb 4A6 as described previously [16 (link), 17 (link)]. The mAb 4A6 was purified from mice ascites fluid by using Protein G Agarose (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Three rounds of biopanning were performed. Then, each well of a 96-well plate was coated with 10 μg/mL mAb 4A6 and blocked with blocking buffer. The phage library was then added to the plate and incubated for 1 h. After five washes with TBS buffer, containing increasing concentrations (0.1%, 0.3%, and 0.5%) of Tween-20, 1 M Tris-HCl was added to the plate [16 (link), 17 (link)]. The eluted bound phages were then amplified and titered on LB/IPTG/Xgal plates for selection. The ratio of output to input was calculated as the titer of the amplified output phages/the titer of the input phages.
7
CAdV-2 Antibody Generation and Phage Display
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The CAdV-2 strain used in this study was stored at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Science. Hybridoma lines secreting CAdV-2-specific mAbs were generated in our lab previously [15 (link)]. The pET-30a (+) vector was purchased from Tiangen, China. A commercially available Ph.D.-12TM Phage Display Peptide Library Kit was purchased from New England BioLabs Inc. CAdV-2 was cultured in Madin–Darby canine kidney (MDCK) cells in Dulbecco’s modified eagle’s medium with 10% fetal calf serum (Gibco, USA) at 37 °C, and Escherichia coli ER2738 and Rosetta (DE3) were cultured in Luria–Bertani (LB) medium.
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