Phage panning was performed to enrich binders to each target protein; immobilized chymotrypsin, and biotinylated KLK1, KLK4, or KLK8. The SPINK2 library was superinfected with the helper phage VCS M13 (Agilent Technologies), following the standard protocol for construction of a phage-display library41 (link). The SPINK2 phage library was cycled through 3–4 rounds of binding selection with each target protein. In the first round, approximately 1.8 × 1013 phages displaying mutated SPINK2 were incubated with each target protein in 3% BSA in PBS containing 0.05% Tween 20 at 4 °C. Phages bound to biotinylated targets were collected using Dynabeads® M-280 Streptavidin (Thermo Fisher Scientific). Bound phages were washed several times with PBS containing 0.05% Tween 20. Then, they were eluted using AcTEV™ Protease (Thermo Fisher Scientific) which cleaved the phages between mutated SPINK2 and gIII proteins. The recovered phage repertoire was amplified in XL1-Blue cells, which were then subjected to the following round of panning. During subsequent selection rounds, the number of washing steps was gradually increased, and the antigen concentration was decreased. For selection of KLK4/8 dual inhibitors, the SPINK2 phage library was subjected to biotinylated KLK4 in round 1, followed by biotinylated KLK4 and KLK8 alternately.
Vcsm13
Manufactured by Agilent Technologies
Sourced in United States
The VCSM13 is a laboratory instrument manufactured by Agilent Technologies. It is designed to perform specific functions within a laboratory setting. No further details can be provided in an unbiased and factual manner without the risk of extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Pbluescript 2, by Agilent Technologies (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Actev protease, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pfu dna polymerase, by Thermo Fisher Scientific (1 mentions)
Oligodeoxynucleotides, by Integrated DNA Technologies (1 mentions)
Microtiter plate reader, by Agilent Technologies (1 mentions)
96 deep well plate, by Corning (1 mentions)
96 well high binding microplates, by Corning (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
6 protocols using vcsm13
1
Phage Panning for Protease Inhibitors
2
Generating Phage Display Libraries
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This protocol was adapted from those to generate phage display libraries(Tonikian et al., 2007 (link)). XL1-Blue MRF’ was transformed with pBluescript II (Agilent 212208). An overnight culture of this strain was prepared in 5 mL LB supplemented with tetracycline (5 μg/ml) and carbenicillin (50 μg/ml) and subcultured the following day 1-in-100 into 5 mL 2YT supplemented with the same antibiotics. At an OD600 of 0.8, cells were infected with helper phage M13KO7 (NEB N0315S) or VCSM13 (Agilent 200251) at a multiplicity of infection of approximately 10-to-1 for 1 h at 37°C The infected cells were used to seed 2YT supplemented with carbenicillin (100 μg/ml) and kanamycin (25 μg/ml) at 1-in-100, and the culture was grown overnight to produce phage. Cells were pelleted at 10,000 g for 15 min, and the supernatant containing phage was transferred. Phage were precipitated by adding 0.2 volumes phage precipitation solution, inverting to mix well, and incubating for 30 min on ice. Phage were pelleted at 15,000 g for 15 min at 4°C and the supernatant was discarded. The phage pellet was resuspended in PBS at 1%–4% of the culture volume. The resuspension was centrifuged to pellet insoluble material and transferred to a new tube. Glycerol was added to a final concentration of 10%–15%. Phage preparations were aliquoted into cryovials and stored at −80°C.
3
Phagemid Vectors for Phage Display
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The construction of phagemid pEB30 for full-length p3 display is described in Huovinen et al. [13 (link)]. The construction of phagemid pEB32x for truncated p3 display, and screening vectors pLK01 and pLK06 are described in Huovinen et al. [14 (link)]. Phagemid pEB92 was constructed by replacing the gene for truncated p3 in vector pAK200 [22 (link)] with p9 amplified from the helper phage VCS-M13 (Agilent Technologies, Santa Clara, CA). Myc tag and Gly2SerGly2 linker preceding the p9 gene were introduced by PCR primers. Tet-cassette-p9 was cloned forward into pEB30 with BamHI and HindIII sites replacing the full-length p3 gene. All restriction enzymes in this work were purchased either from New England Biolabs (Ipswich, UK) or from Thermo Scientific (Waltham, MA, USA). Ligations were carried out with T4 DNA ligase and PCR either with Pfu DNA polymerase or Phusion DNA polymerase, which were all purchased from Thermo Scientific (Waltham, MA, USA). Purification of PCR products and gel extraction were performed with kits from Qiagen (Hamburg, Germany), whereas minipreps were prepared with a kit from Thermo Scientific (Waltham, MA, USA).
4
Generating Phage Display Libraries
5
Oligodeoxynucleotide-based PCR Primer Sourcing
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The oligodeoxynucleotides used as PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, USA), and Hokkaido System Science (Sapporo, Japan) in purified forms. VCSM13 was obtained from Agilent Technologies (Santa Clara, CA, USA). The 1 kbp ladder DNA size marker was from GeneDireX (Taipei, Taiwan).
6
Phage ELISA for CADM1 Binding
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Single colonies were inoculated into 1 mL of SB media containing 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C overnight. Then, 1012 pfu/mL helper phages (VCSM13; Agilent, Santa Clara, CA, USA) and 70 μg/mL of kanamycin were added and incubated overnight at 37 °C. The plates were centrifuged at 4000 rpm, and the supernatant was applied for phage ELISA. The 96-well high-binding microplates (Corning, Corning, NY, USA) were coated overnight with 0.1 μg of MF-hCADM1 and MF-mCADM1 in phosphate-buffered saline (PBS) at 4 °C. After blocking with 3% (w/v) BSA in PBS, the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After washing three times with PBS containing 0.05% (v/v) tween 20 (PBST), HRP-conjugated anti-hemagglutinin (HA) antibody (1:3000; Bethyl Laboratories, Montgomery, TX, US) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed by adding 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) as the chromogenic substrate. The reactions were stopped by the addition of 2 M of sulfuric acid (H2SO4), and the absorbance was read at 450 nm using a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA).
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