Ligand blotting was performed according to the protocol reported by Fernandez-Luna et al [26 (link)]. Total C. elegans proteins were separated by 2-DE, and gels were transferred to a PVDF membrane in transfer buffer (20% methanol, 25 mM Tris-base, 192 mM glycine) for 60 min. Filters were blocked overnight in PBST (0.1% Tween 20 in phosphate buffered saline, pH 7.4) containing 3% BSA. Blocked filters were incubated with biotinylated Cry6Aa (10 nM) for 2 h at room temperature, and then washed three times using PBST. The bound protein was detected by 1 μg/ml streptavidin- horseradish peroxidase (HRP) conjugate (Sigma, S5512). The membrane was visualized using enhanced chemiluminescence substrate (SuperSignal West Pico, Pierce) following the manufacturer’s instructions.
Streptavidin horseradish peroxidase hrp conjugate
Manufactured by Merck Group
Sourced in United States
Streptavidin–horseradish peroxidase (HRP) conjugate is a laboratory reagent that consists of the protein streptavidin covalently linked to the enzyme horseradish peroxidase. It is commonly used in various biochemical and immunoassay techniques where the detection of biotinylated molecules is required.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Biotinylated anti human ige monoclonal antibody, by Vector Laboratories (2 mentions)
Labsystems multiskan, by Thermo Fisher Scientific (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Chelerythrine chloride, by Merck Group (1 mentions)
Donkey anti mouse igg hrp ab205724, by Abcam (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
Phospho p38 mapk thr180 tyr182 antibody 9211, by Cell Signaling Technology (1 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (1 mentions)
Sb203580 5633, by Cell Signaling Technology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Thermo Fisher Scientific (1 mentions)
Roti histofix 4, by Carl Roth (1 mentions)
Amersham hyperfilm ecl, by Cytiva (1 mentions)
9 protocols using streptavidin horseradish peroxidase hrp conjugate
1
Ligand Blotting of C. elegans Proteins
2
Sandwich ELISA for HCV Core Antigen Quantification
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The sandwich ELISA was performed using the streptavidin-biotin peroxidase complex (ABC) assay. Briefly, wells were coated overnight at room temperature with 100 µL of 5 µg/mL mouse monoclonal antibody against 21 - 40 aa of HCV core antigen (Abcam Co., UK) and blocked with 1% BSA for two hours at 37°C. After several washing steps, 50 μg of TSP from each plant extract was added to the coated wells in duplicates and incubated for one hour at 37°C. Following washing steps, biotinylated anti-core polyclonal antibodies (1:1000 dilution; Abcam Co., UK) were immersed into the wells and incubation continued at 37°C for one hour followed by washing steps and another similar incubation with streptavidin horseradish peroxidase (HRP) conjugate (1:4000; Sigma, USA). Finally, by adding TMB and color development, the absorbance was measured at 450 nm.
To quantify the assay, a standard curve was prepared by applying serial dilution (in 0.1 - 1µg/ml range) of purified bacterial HCVcp to determine HCVcp expression level in seeds. The E. coli-derived HCVcp (eHCVcp) was purified through application of Nickel-Nitrilotriacetic Acid (Ni-NTA) chromatography as previously described (12 (link), 35 (link)).
To quantify the assay, a standard curve was prepared by applying serial dilution (in 0.1 - 1µg/ml range) of purified bacterial HCVcp to determine HCVcp expression level in seeds. The E. coli-derived HCVcp (eHCVcp) was purified through application of Nickel-Nitrilotriacetic Acid (Ni-NTA) chromatography as previously described (12 (link), 35 (link)).
3
ELISA for S-ED Protein Quantification
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Ninety-six-well plates were coated with 100 μL per well of 100 ng of rabbit polyclonal anti-S-ED serum in 50 mM carbonate/bicarbonate buffer (pH 9.6) and incubated overnight (ON) at 4 °C. After blocking 1 h at 37 °C with 2% (w/v) non-fat milk in PBS, plates were incubated with 1:2 serial dilutions of S-ED internal standard or samples (culture supernatants, harvests, different purification fractions) for 1 h at 37 °C. At the same time, the following controls were tested in each run: (1) a reactive blank and (2) a sample blank. Then, plates were incubated with biotinylated rabbit anti-S-ED antibodies diluted 1:1500 for 1 h at 37 °C. Afterwards, streptavidin–horseradish peroxidase (HRP) conjugate (Sigma) diluted 1:2.000 was added to the wells. Finally, plates were incubated for 13 min with substrate solution (0.5 mg.mL−1 o-phenylenediamine, 0.5 μl.mL−1 H2O2 30 vol in 50 mM phosphate citrate buffer). Absorbance was measured at 492 nm with a microtiter plate reader (LabSystems Multiskan, Thermo Fisher Scientific). Between every step, plates were washed six times with 0.05% (v/v) Tween-20 in PBS (PBS-T). Dilutions of tested samples and antibodies were prepared in PBS-T containing 0.2% (w/v) non-fat milk.
4
Cry6Aa Binding Assay on Nitrocellulose
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Different quantities of ASP-1 were dotted onto a nitrocellulose (NC) membrane (Millipore, Bedford, MA, USA). After blocking with 3% bovine serum albumin (BSA) in PBST, NC membrane was bathed in biotinylated-Cry6Aa (10 nM) for 2 h at room temperature, washed three times using PBST. Unlabeled Cry6Aa (1000-fold excess) was used in the competition assays. The bound protein was detected by 1 μg/ml streptavidin-horseradish peroxidase (HRP) conjugate (Sigma, S5512). Finally, the signal was visualized using 3, 3'-diaminobenzidine tetrahydrochloride (DAB) substrate (Pierce) following the manufacturer’s instructions.
5
Amyloid-β Peptide Binding and Signaling
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Most of the material used in this study was purchased as we reported earlier [4 (link),53 (link),68 (link),96 (link),97 (link),98 (link),99 (link),100 (link)]. Aβ 1–40 (AS-24235), Aβ 1–42 (AS-20276), Aβ 1–28 (AS-24231), Aβ 25–35 (AS-24227), and Aβ 1–16 (AS-24225) were purchased from AnaSpec. Biotin-HN (HN, B-018--26, UniProt Q8IVG9) was purchased from Phoenix Pharmaceuticals. Phosphate Buffered Saline (PBS), nitrocellulose membranes, streptavidin–horseradish peroxidase (HRP) conjugate, Ponceau S solution, chelerythrine chloride, and ACh were purchased from Sigma-Aldrich. Mouse α-tubulin monoclonal antibody (DM1A), goat anti-rabbit IgG (H+L) secondary antibody (HRP, 31466), 3,3′,5,5′-tetramethylbenzidine (TMB), BCA protein assay kit, super signal west pico luminol (chemiluminescence) reagent, lipofectamine 2000 transfection reagent, and the Halt protease and phosphatase inhibitor cocktail were from ThermoFisher. Donkey anti-mouse IgG (HRP) (ab205724) was purchased from Abcam. m-IgGκ BP-HRP was obtained from Santa Cruz Biotechnology. SignalSilence p53 siRNA I (6231), SignalSilence control siRNA (Unconjugated, 6568), rabbit p53 antibody (9282), p38 MAPK antibody (9212) that detects endogenous levels of total p38α/β/γ MAPK, phospho-p38 MAPK (Thr180/Tyr182) antibody (9211), PKCα antibody (2056), and SB203580 (5633S) were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Streptavidin-HRP Detection of Labeled Proteins
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Labeled protein extracts (10–15 μg protein) were mixed with 4× lithium dodecyl sulfate (LDS) gel loading dye [423 mM Tris-HCl, 563 mM Tris base, 8% (w/v) LDS, 40% (w/v) glycerol, 2 mM EDTA, 0.075% (w/v) SERVA Blue G250; freshly supplemented with 100 mM DTT], incubated at 70°C for 15 min and separated by denaturing polyacrylamide gel electrophoresis on 11% Bis-Tris resolving gels. The separated proteins were transferred on a PVDF membrane (Merck, United States) using a tank blot setup (Bio-Rad, United States) and the membrane was washed thrice with TBS-T [20 mM Tris base pH 7.5, 150 mM NaCl, 0.2% (w/v) Tween20]. Blocking with 3% BSA (w/v) in TBS-T was done overnight at 4°C, followed by incubation with a streptavidin-horse radish peroxidase (HRP) conjugate (Sigma-Aldrich, United States) directly added into the blocking solution (1:25,000) for 2.5 h at room temperature. The membrane was washed 6× with TBS-T and the labeled proteins were detected by enhanced chemiluminescence (ECL) with a mix of the SuperSignal® West Pico Chemiluminescent Substrate and the SuperSignal® West Femto Maximum Sensitivity Substrate (4:1; Thermo Scientific, United States) using an Amersham Imager 600 (GE Healthcare Life Sciences, Germany).
7
Immunoblotting Analysis of Allergen-Specific IgE
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Purified P34 and MBP (3 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocked membranes (3% horse serum) were incubated with patient serum (1:5, overnight at 4℃), followed by biotinylated anti-human IgE monoclonal antibody (1:3,000, 4 hours at 37℃, Vector Laboratories Inc., CA, USA), and finally, with horseradish peroxidase (HRP) -streptavidin conjugate (1:3,000, 30 minutes at 37℃, Sigma-Aldrich, MO, USA). Luminol chemiluminescent substrate and exposure to X-ray film (Amersham Hyperfilm ECL, GE Healthcare Bio-Sciences Corp., USA) were used for band visualization. Membranes were also revealed using the mAbs (1 µg/mL) followed by mouse immunoglobulin G-specific rat monoclonal antibody HRP-conjugate (1:3,000, 1 hour at 37℃ Bio-Rad Laboratories, CA, USA).
8
Quantification of Adipose Tissue Hyaluronan
9
Proteomic Analysis of Allergen Extracts
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Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of SE was performed as previously described [28] .
Briefly, for the first dimension, 80 g SE were applied using immobilized pH gradient 7 cm strips (pH 3-10); and for the second dimension, the strips were run on 12.5% (w/v) SDS-PAGE gels. Gels were stained with Coomassie blue G250. 2D-PAGE was transferred to nitrocellulose membrane and blocked with 3% horse serum in PBS. Blocked membranes were incubated with a pool of patient sera (1:5, overnight at 4ЊC), followed by biotinylated anti-human IgE monoclonal antibody (1:3000, 4 h at 37ЊC, Vector Laboratories Inc., CA, USA), and horseradish peroxidase (HRP)-streptavidin conjugate (1:3000, 30 min at 37ЊC, Sigma-Aldrich, MO, USA). Luminol chemiluminescent substrate and exposure to X-ray film (Amersham Hyperfilm ECL, GE Healthcare Bio-Sciences Corp., USA) were used for spot visualization. The protein identification of immunoreactive spots was determined by PMF MALDI-TOF MS [29] . Search parameters were set as described elsewhere (MASCOT 2.6.0, database: SwissProt 2016_10, enzyme: trypsin; variable modifications: carbamidomethyl (Cys), oxidation (Met), peptide mass tolerance: 35 ppm, taxonomy: other green plants and max missed cleavages: 2).
Briefly, for the first dimension, 80 g SE were applied using immobilized pH gradient 7 cm strips (pH 3-10); and for the second dimension, the strips were run on 12.5% (w/v) SDS-PAGE gels. Gels were stained with Coomassie blue G250. 2D-PAGE was transferred to nitrocellulose membrane and blocked with 3% horse serum in PBS. Blocked membranes were incubated with a pool of patient sera (1:5, overnight at 4ЊC), followed by biotinylated anti-human IgE monoclonal antibody (1:3000, 4 h at 37ЊC, Vector Laboratories Inc., CA, USA), and horseradish peroxidase (HRP)-streptavidin conjugate (1:3000, 30 min at 37ЊC, Sigma-Aldrich, MO, USA). Luminol chemiluminescent substrate and exposure to X-ray film (Amersham Hyperfilm ECL, GE Healthcare Bio-Sciences Corp., USA) were used for spot visualization. The protein identification of immunoreactive spots was determined by PMF MALDI-TOF MS [29] . Search parameters were set as described elsewhere (MASCOT 2.6.0, database: SwissProt 2016_10, enzyme: trypsin; variable modifications: carbamidomethyl (Cys), oxidation (Met), peptide mass tolerance: 35 ppm, taxonomy: other green plants and max missed cleavages: 2).
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