Ninety-six-well plates (Greiner Bio-One, Frickenhausen, Germany) were pre-coated with rat anti-mouse C5a capture antibody (4 μg/mL; R&D Systems, Minneapolis, MN, USA) diluted in PBS (pH 7.4) overnight at room temperature in a sealed humidified container. Following the plate being blocked for 1 h at room temperature with reagent diluent (1% BSA in PBS), C5a standard, spinal cord and TA muscle homogenates were incubated for 2 h at room temperature. The plates were subsequently incubated with biotinylated goat anti-mouse C5a detection antibody (0.2 μg/mL, R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature, and then incubated with Streptavidin-HRP conjugate for 20 min at room temperature. Tetramethylbenzidine (Sigma-Aldrich, Saint Louis, MO, USA) substrate was used as the chromogen, and the plate was read at 450 nm. Levels of C5a in spinal cord and TA muscle samples were adjusted to micrograms per protein and expressed as nanograms of C5a per microgram of protein.
Streptavidin hrp conjugate
Manufactured by R&D Systems
Sourced in United States
Streptavidin-HRP conjugate is a laboratory reagent composed of streptavidin, a protein derived from the bacterium Streptomyces avidinii, and horseradish peroxidase (HRP), an enzyme commonly used as a reporter molecule in various assays. The streptavidin-HRP conjugate is designed to bind to biotinylated targets, enabling the detection and visualization of these targets through HRP-catalyzed colorimetric or chemiluminescent reactions.
Automatically generated - may contain errors
Lab products found in correlation
Tetramethylbenzidine, by Merck Group (1 mentions)
Ninety six well plates, by Greiner (1 mentions)
Pierce classic ip kit, by Thermo Fisher Scientific (1 mentions)
Su6656, by Merck Group (1 mentions)
Recombinant vegfa, by R&D Systems (1 mentions)
Primary human umbilical vein endothelial cells huvecs, by Thermo Fisher Scientific (1 mentions)
Hrp substrate, by R&D Systems (1 mentions)
Matrigel matrix growth factor reduced, by BD (1 mentions)
Biotinylated il 17a, by R&D Systems (1 mentions)
Spectramax m5e microplate reader, by Molecular Devices (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Bm blue pod, by Roche (1 mentions)
Bio plex manager 6, by Bio-Rad (1 mentions)
Recombinant murine il 4, by R&D Systems (1 mentions)
Bvd6 24g2, by Thermo Fisher Scientific (1 mentions)
Abts substrate, by Roche (1 mentions)
Goat anti rabbit igg hrp conjugate, by Merck Group (1 mentions)
5 protocols using streptavidin hrp conjugate
1
Quantification of C5a in Spinal Cord and Muscle Tissues
2
Exploring VEGFR-2 and Integrin Signaling in HUVECs
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Primary human umbilical vein endothelial cells (HUVECs) were purchased from Cascade Biologics. FN, MGO and su6656 were obtained from Sigma. Recombinant VEGF‐A, recombinant RAGE, recombinant human integrin α5β1 protein, RAGE function‐blocking antibody, biotinylated anti‐RAGE antibody, biotinylated anti‐integrin β1 antibody, streptavidin‐HRP conjugate and HRP substrate were from R&D Systems. Growth‐factor‐reduced Matrigel Matrix was from BD Biosciences. Antibodies to phosphorylated VEGFR‐2, total VEGFR‐2, phosphorylated Akt, total Akt, phosphorylated extracellular regulated protein kinases 1/2 (ERK1/2), total ERK1/2, phosphorylated nuclear factor‐κB (NF‐κB), total NF‐κB and CD31 were from Cell Signaling Technology. Antibodies to FN and AGEs were from Abcam. Antibodies to RAGE and c‐Src were from Santa Cruz Biotechnology. Pierce classic IP kit was from Thermo Scientific.
3
IL-17A Receptor Binding Inhibition Assay
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All four 2C1-Fc fusion proteins were tested in ELISA competition experiments to determine their capabilities to block the interaction between IL-17A and its receptor. Maxisorp plates (Nunc) were coated with commercially available IL-17R-Fc fusion (R&D Systems) overnight at a concentration of 0.6 μg/ml. After blocking with 1% BSA in PBS, different concentrations of the Fc fusions were pre-incubated with 2.5 nm of biotinylated IL-17A (R&D Systems; biotinylated in-house with NHS-PEG4-biotin according to the manufacturer's instructions (Pierce)); and added to the wells. After washing, the wells were incubated with streptavidin-HRP conjugate (R&D Systems) 1:200 diluted in 1% BSA/PBS. After washing, BM Blue POD (Roche Applied Science) was added as substrate for color development. This reaction was stopped after 5 min with 1 m sulfuric acid (Sigma). For each condition, the reference-subtracted absorbance was calculated by A450 − A650 (reference wavelength) using a SpectraMax® M5e Microplate Reader (Molecular Devices). IC50 values were calculated using Prism software (version 5).
4
Quantification of Murine IL-4 and TGFβ
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IL-4 levels in culture supernatants were determined by a sandwich ELISA specific for murine IL-4 using paired capture and detection antibodies (clone 11B11 and biotin-conjugated clone BVD6-24G2; eBioscience) and developed by streptavidin-HRP conjugate (R&D Systems, Minneapolis, MN) and ABTS substrate (Roche, Indianapolis, IN) as previously described [42 (link)]. Cytokine concentrations were calculated based on a standard curve generated using serial dilutions of murine recombinant IL-4 (R&D Systems). In some experiments, cytokine levels were determined in supernatants of AWH-stimulated MLN cells or serum using a custom-made cytokine Bio-Plex Pro 10-Plex assay (Bio-Rad, Hercules CA) according to the manufacturer’s instructions. Acquisition was performed on the MAGPIX platform (Luminex) and data were analyzed using the Bio-Plex Manager 6.1 software (Bio-Rad). To determine TGFβ levels, supernatants were acid-activated and neutralized prior to assay using an ELISA performed according to the manufacturer’s instructions (Mouse TGFβ DuoSet, R&D Systems).
5
Quantitative ELISA for HER2 Protein
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All the reagents and buffer components were purchased from Sigma-Aldrich, unless otherwise specified. Bi-distilled (dd-H2O) or Milli-Q grade water was used. Mouse monoclonal antibodies (IgG mAbs) MGR2 and MGR3 directed against the extracellular domain of HER2, obtained and purified as previously described20 (link) were stored sterile at 4 °C in a buffer containing 150 mM sodium chloride and 0.02% sodium azide at pH 7.4.
Recombinant HER2 ECD protein (code 10004-HCCH, 70 kDa), and rabbit anti-human HER2 polyclonal antibody (IgG pAb, code 10004-RP04) were obtained from Sino Biological Inc. and stored in multiple aliquots at −20 °C. Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively. Goat anti-rabbit IgG-HRP conjugate (1 mg mL−1, code 401353) and 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate system for ELISA (code ES001) were purchased from Merck Millipore and stored at 4 °C together with lyophilized human serum albumin (HSA, code A1653, Sigma-Aldrich).
Herceptin (trastuzumab 150 mg, Roche) was kindly provided by Dr. Renzo Lazzarini, Pharmaceutical Division, CRO Aviano, resuspended and stored as recommended by the manufacturer.
Recombinant HER2 ECD protein (code 10004-HCCH, 70 kDa), and rabbit anti-human HER2 polyclonal antibody (IgG pAb, code 10004-RP04) were obtained from Sino Biological Inc. and stored in multiple aliquots at −20 °C. Goat anti human HER2 biotinylated polyclonal antibody (IgG pAb-biotin, code BAF1129) and Streptavidin-HRP conjugate (code DY998 Part #890803 5 vials) were from R&D Systems and stored at −20 °C and at 4 °C respectively. Goat anti-rabbit IgG-HRP conjugate (1 mg mL−1, code 401353) and 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate system for ELISA (code ES001) were purchased from Merck Millipore and stored at 4 °C together with lyophilized human serum albumin (HSA, code A1653, Sigma-Aldrich).
Herceptin (trastuzumab 150 mg, Roche) was kindly provided by Dr. Renzo Lazzarini, Pharmaceutical Division, CRO Aviano, resuspended and stored as recommended by the manufacturer.
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