Pools of purified phage from the library stock (Sheets), and the Round 1, Round 2, Round 3 and Round 4 amplified stocks from the CD83-GFP biopanning were tested for binding to soluble CD83 using ELISA. Nunc Maxisorp 96-well plates were coated with 5 μg/mL CD83 in PBS overnight at room temperature. The wells were washed three times with PBS and then blocked for 1 hr with MPBS. Phage pools (~1011 phage particles) were also blocked in MPBS. Dilutions of the blocked phage were then added to the CD83 coated plate and incubated for 1 hr at room temperature. The wells were washed three times with PBS containing 0.1% (v/v) Tween-20 (PBST), and then 200 μL secondary antibody added (1/5000 dilution of HRP/Anti-M13 Monoclonal Conjugate-GE Healthcare) and incubated for 1 hr at room temperature. The wells were washed three times with PBST, followed by addition of 100 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich). The reaction was stopped after 10 mins using 100 μL 1 M sulphuric acid. Absorbance was measured at 450 nm using BioTek Powerwave HT Microplate Reader (Millenium Science).
Hrp anti m13 monoclonal conjugate
Manufactured by GE Healthcare
The HRP/Anti-M13 Monoclonal Conjugate is a laboratory reagent used for detection and quantification of M13 bacteriophage in various assays. It consists of horseradish peroxidase (HRP) enzyme conjugated to a monoclonal antibody specific for the M13 phage. This conjugate can be utilized in immunoassay techniques to identify and measure the presence of M13 phage in samples.
Automatically generated - may contain errors
Lab products found in correlation
Tmb liquid substrate, by Merck Group (2 mentions)
Thrombin cleavage capture kit, by Merck Group (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Bugbuster master mix, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Maxisorp microplate, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
4 protocols using hrp anti m13 monoclonal conjugate
1
Phage Library Screening for CD83 Binding
2
Recombinant Scaffold Protein Expression
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Gene-of-interests including scaffold candidates and enriched phagemid DNA were cloned into the pET32a vector. Scaffold candidates were as follows; Kunitz-type inhibitors, collagen IV α3 chain C5 domain (UniProt; P12111, 2501–2558) and HGF activator inhibitor-1 (UniProt; O43278, 245–303); Kazal-type inhibitors, SPINK2 (UniProt; P20155, 23–84) and LEKTI 15th domain (UniProt; Q9NQ38, 989–1064); Epiregulin (UniProt; O14944, 63–108); β-defensin-1 (UniProt; P60022, 33–68). Origami B (DE3) were transformed with each expression vector and cultured at 37 °C, then IPTG was added. After cultivation at 16 °C overnight, cell pellets were collected and lysed using BugBuster® Master Mix (EMD Millipore). Protein-of-interests were purified using TALON® Metal Affinity Resins (Clontech), then thioredoxin tags were removed using a Thrombin Cleavage Capture Kit (EMD Millipore). They were finally purified using Superdex-75 (GE Healthcare) size exclusion chromatography, and applied to enzyme assays for binder selection, as described below. Multimeric formation analysis of candidates was confirmed by SDS-PAGE, followed by western blotting analysis: goat anti-S-Tag Antibody HRP Conjugated (Betyl) was used for purified proteins; HRP/Anti-M13 Monoclonal Conjugate (GE healthcare) was used for detection of proteins fused to the bacteriophage M13 gIII protein.
3
Binding of Tox1 and Tox2 to eEF2 by ELISA
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Example 3
Binding of Tox 1 and Tox2 to eEF2 was tested by ELISA using eEF2 or BSA as ligands.
Experimental Part
0.25 μg of target proteins, eEF2 (Human; Yeast derived) or BSA (negative control) were applied to several wells of maxisorp plate (NUNC) in 50 μl PBS and incubated over night at 4° C. The solutions were removed, and each well was supplemented with 280 μl blocking solution (BSA 2 mg/ml). The plate was incubated 1 hr at 25° C.
To 1.5 ml tubes 100 μl of blocking solution+109 pfu (plaque forming units) of M13 phages that express the following peptides: eEF2-binding: RB, LBR1, TB2 (Tox2), YO2, GW (Tox1), DRB, PY, BW were added. The plate was incubated 1 hr at 25° C. The solution were discarded and the wells were washed 7 times with 280 μl washing solution (Tween 20 0.05%).
Each well was supplemented with 50 μl of HRP/Anti-M13 Monoclonal Conjugate (GE Healthcare) diluted 1:5000. The plate was incubated 1 hr at 25° C. The solutions were discarded and the wells were washed 7 times with 280 μl washing solution (Tween 20 0.05%). Each well was supplemented with 50 μl of TMB (T0440; Sigma).
The plate was photographed using a scanner after incubation time of 1.5 min and 30 min. It can be seen from
4
Capture and Detection of CD1b and CD1e Complexes
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A 96-well Maxisorp microplate (Nunc, United States) was coated (16 h/4°C) with: (a) the anti-CD1b mAb BCD1b3.1 (Sigma, United States) to capture CD1b complexes and free CD1b; (b) the anti-CD1e mAb CD1e20.6 to capture the recombinant human CD1e. Both mAbs were diluted in carbonate-bicarbonate pH 9.6 coating buffer (3 μg/mL). The plate was then blocked with 3% PBS-skim milk (1 h/RT). Two washes (PBS-tween-20, 0.05%) were performed, and the complexes and free CD1b were added in PBS pH7.4 (1 μg/mL). After three washes (PBS-tween20, 0.05%), scTCR and dAbκ11, diluted in 3% PBS-skim milk (108 phages) were added and the plate was incubated for 1 h at RT. Subsequently, three washes were performed, and HRP/Anti-M13 monoclonal conjugate (GE, Healthcare, Life Sciences; diluted 1:5000 in 3% PBS-skim milk) was added. 1 h later, the plate was washed as above, TMB liquid substrate (Sigma, United States) was added, followed by incubation (10 min/RT). The reaction was stopped with 1 M sulfuric acid (Merck, Germany). Absorbance values (450 nm) were measured in a microplate reader (BioRad, United States). Each sample was studied in triplicate, and the experiment was repeated three times.
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