Different concentrations of the purified recombinant sdAb were added to a 10 μg/ml S protein-coated 96-well Stripwell microplate (Corning, USA), and the protein extracted from the bacterial cells transformed with the empty pET-25b vector containing both an SBP-tag and a 6× His tag was used as a negative control. PBS was used as a blank control. The binding activity of each well was detected with HRP-streptavidin (Solarbio Life Sciences, Beijing, China) at a 1:10,000 concentration and visualized with the TMB solution, and the plate was read at 450 nm in a microplate reader.
Hrp streptavidin
Manufactured by Solarbio
Sourced in China
HRP-streptavidin is a conjugate of horseradish peroxidase (HRP) and streptavidin. Streptavidin is a protein derived from the bacterium Streptomyces avidinii, known for its strong and specific binding to biotin. The HRP enzyme is commonly used as a reporter or label in various immunoassay and detection techniques.
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Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Transetta de3, by Transgene (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
α lactalbumin, by Merck Group (1 mentions)
β lactoglobulin, by Merck Group (1 mentions)
Igg2a hrp, by Southern Biotech (1 mentions)
Tmb 1 component peroxidase substrate, by Thermo Fisher Scientific (1 mentions)
Synergy htx instrument, by Agilent Technologies (1 mentions)
Igg1 hrp, by Southern Biotech (1 mentions)
Igg3 hrp, by Southern Biotech (1 mentions)
Biotinylated nad, by Bio-Techne (1 mentions)
Anti his antibody, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using hrp streptavidin
1
Quantifying recombinant sdAb binding
2
Isolation and Purification of sdAb
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The plasmids of the positive clones from phage ELISA were isolated, and the VHH gene was digested with NcoI and NotI and ligated to a modified pET-25b vector that contained the 38-amino acid sequence of streptavidin binding protein (SBP) between the NotI and XhoI restriction sites (Additional file 1 : Fig. S1) The resulting vector was transformed into competent E. coli Transetta-DE3 (Transgene, Beijing, China). Expression of the recombinant sdAb was induced by 1 mM IPTG, and then the proteins were purified by the Ni–NTA Agarose (Qiagen, Germany) under native conditions. The expression and purification of the sdAb was analyzed by SDS-PAGE electroporation and western blot with HRP-streptavidin (Solarbio Life Sciences, Beijing, China).
3
Comparative Analysis of BMP-Biotin Linkage
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The linkage rate of BMP−∆F−BF and BMP−biotin with HRP−streptavidin was compared with commercial biotin-labeled magnetic beads (MB−biotin; superparamagnetic iron oxide; particle size 20–50 nm; Nanocs, Natick, MA, USA). BMP−∆F−BF, BMP−biotin, and MB−biotin were incubated with 1% bovine serum albumin (BSA) and stored at 4 °C. One−step ELISA was used to detect the linkage rate. A 96−well microtiter plate coated with 300 μL 2% BSA was incubated at 4 °C overnight and washed thrice with 250 μL 10 mM PBST (pH 7.4). Then, 2 mg BMP−∆F−BF, BMP−biotin, and MB−biotin each were added (each BMPs and MB three parallel) and captured using a magnetic rack. The sample was then washed thrice with PBST, and 100 μL 1:2000 diluted HRP-streptavidin (Beijing Solarbio Science & Technology, Beijing, China) was added to each well, with coupling for 1 h at 600 rpm at room temperature, captured by a magnetic rack and washed five times with PBST. The color was developed using 100 μL tetramethylbenzidine (TMB) for each well, reacted for 3 min at room temperature, and then 50 μL 2 M H2SO4 was added to stop the reaction. The supernatant was transferred to a new 96−well microtiter. Absorbance at 450 nm was measured on a microplate reader (blank control, only BMPs and MB incubated, no HRP-streptavidin). The OD450 value was calculated to compare the BMPs and MB linkage rates.
4
Assessing IgE Binding Capacity in WPI
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The capacity of both α-LA and β-LG in WPI to bind specific IgE was assessed by indirect competitive ELISA according to the method of Liu et al. [23 (link)] with slight modifications. α-Lactalbumin (L6010, purity 85%, Merck KGaA, Darmstadt, Germany) and β-lactoglobulin (L3908, purity 90%, Merck KGaA, Darmstadt, Germany) were dissolved in carbonate buffer solution (CBS, 0.05 mol/L, pH 9.6) at a concentration of 50 μg/mL and coated in ELISA plates, respectively, and serum diluted 20 times. Goat anti-human IgE antibody, biotin conjugate (Thermo Fisher Scientific, Shanghai, China) was dissolved in phosphate-buffered saline (PBS, 0.1 mol/L, pH 7.4) at a ratio of 1:2000. HRP-streptavidin (Solarbio, Beijing, China) was dissolved in PBS at a ratio of 1:2000.
OD values at 450 nm and 630 nm were measured on a microplate reader (Thermo Scientific, Massachusetts, USA). The inhibition rate was calculated according to the following formula:
ΔOD: The result of ELISA was the difference in the optical density of samples.
ΔOD0: The difference in optical density of noncompetitive.
OD values at 450 nm and 630 nm were measured on a microplate reader (Thermo Scientific, Massachusetts, USA). The inhibition rate was calculated according to the following formula:
ΔOD: The result of ELISA was the difference in the optical density of samples.
ΔOD0: The difference in optical density of noncompetitive.
5
ELISA Assay for S-Protein Antibody Titers
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S-protein-specific serum antibody titers were measured using ELISA. Corning ELISA plates were incubated with 10 µg/mL of the recombinant S protein purified in our lab in phosphate-buffered saline (PBS) overnight at 4 °C. After three washes, the plates were blocked with 1 × ELISAPOT Diluent (Invitrogen, cat. 00-4202-56, Waltham, MA, USA) for 2 h at 25 °C. Following another washing, the plates were incubated overnight using mouse serum at 4 °C and diluted with PBST. Following a set of washings, the plates were incubated with a 1:5000 dilution of goat anti-mouse IgG (H + L)-BIOT, IgG1-HRP, IgG2a-HRP or IgG3-HRP (Southern Biotech, cat. 1036-08, 1071-05, 1081-05 and 1101-05) for 1 h at room temperature. For IgG (H + L)-BIOT, following three washes, the plates were incubated with streptavidin-HRP (Solarbio, cat. SE068, Beijing, China) for 30 min at 25 °C. Finally, after washing the plates, the reaction was revealed using TMB 1-Component Peroxidase Substrate (Invitrogen, cat. 00-4201-56) and impeded using a 2 M HCl solution. The absorbance at 450 nm was determined within 30 min using a Synergy HTX instrument (BioTek Instruments, Highland Park, VT, USA).
6
Biotinylated NAD+ Protein Interactome
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The purified proteins were blotted onto the methanol-activated PVDF membrane (Millipore, USA). After drying at room temperature, the membranes were incubated with 25 μM biotinylated NAD+ (Trevigen, USA) and 500 nM synthesized DNA oligos for 30 min at 25 °C. The blot was washed with TBST buffer (50 mM Tris, 0.5 M NaCl, and 0.05% Tween-20, pH 8.0) and then incubated with streptavidin/HRP (Solarbio, China) at room temperature for 2 h. After reaction with chemiluminescent substrates, fluorescence signals were detected using a ChemScope 3300 mini instrument (CLINX, China). BSA and tag proteins were used as negative controls, and AtPARP1 was used as the positive control. The signals obtained with anti-His antibody (Proteintech, USA) were used as the protein loading control for each spot.
7
Photocatalytic Protein Labeling Protocols
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Photocatalyst (5 μM) was combined with PAB-caged QM/thioQM probes (100 μM), NADH (500 μM), and bovine serum albumin (BSA, 1 mg/mL) in PBS (pH 7.4) to afford reaction mixtures with total solution volume of 100 µL. The samples were placed on a 96-well plate (100 μL/well) and irradiated by mild blue LED (450 nm, 4 mW/cm2) (unless otherwise noted) at room temperature for 5 min (unless otherwise noted). Eighty microliter of each sample was taken and combined with 20 µL 5× SDS-PAGE non-reducing loading buffer (Beyotime). Immunoblotting was performed with 4–15% gradient SDS-PAGE and 0.2 μm PVDF membrane. Streptavidin-HRP (Solarbio, SE068, 1:1,000 dilution) was used to detect the biotinylated bands. For validation of photocatalytic labeling by SF2 , groups without Ir1 or without light irradiation were used as controls.
For UV-triggered labeling,SF2 UV (100 μM) was combined with BSA (1 mg/mL) in PBS (pH 7.4) to afford reaction mixtures with a total solution volume of 100 µL. The samples were placed on a 96-well plate (100 μL/well), irradiated by UV light (CL-1000 UVP Ultraviolet Crosslinker) for 10 min at room temperature, and analyzed by immunoblotting as described above.
For UV-triggered labeling,
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