The equilibrium dissociation constant (Kd) values of ErbB2 aptamer were determined using an enzyme-linked oligonucleotide assay (ELONA). Briefly, 96-well ELISA plates (Corning®, Sigma-Aldrich) were coated with ErbB2 (Cat. No. 1129-ER, R&D system) at 4 ºC for 16 h. The treated wells were blocked with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, followed by three washes with PBS. The biotin-labeled aptamers were added and incubated for 1 h at room temperature. After extensive washing with washing buffer (10 mM PBS, 0.05% Tween-20, pH 7.4), Pierce™ High Sensitivity Streptavidin HRP (Thermo Fisher Scientific) was added to each well to bind biotin-conjugated aptamer. After incubation for 1 h at room temperature, the plates were washed again as described above. Tetramethyl benzidine (TMB) substrate solution (Thermo Fisher Scientific) was added to each well and incubated for 30 min at room temperature. Then, the reaction was quenched by addition of 2 M H2SO4. The protein-bound aptamer-streptavidin complexes were quantified by determining the absorbance at 450 nm using GloMax® Discover System (Promega, Madison, WI, USA). The saturation curve was plotted and Kd was analyzed with Sigma Plot 12.5 (https://systatsoftware.com/products/sigmaplot/ ) software.
Pierce high sensitivity streptavidin hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce™ High Sensitivity Streptavidin-HRP is a laboratory reagent used to detect and quantify biotinylated molecules in various assays. It is a conjugate of the protein streptavidin and the enzyme horseradish peroxidase (HRP). The high sensitivity of this product is due to the strong binding affinity between streptavidin and biotin, enabling the detection of low-abundance biotinylated targets.
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Lab products found in correlation
Pierce protein free pbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Tetramethylbenzidine tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Glomax discover system, by Promega (1 mentions)
Tmb substrate reagent, by BD (1 mentions)
Elisa plates, by Iwaki (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Immun blot polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Tween 20, by Nacalai Tesque (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon p polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemidoc gel documentation system, by Bio-Rad (1 mentions)
Hrp conjugated anti mouse and anti rabbit secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Precision plus protein kaleidoscope, by Bio-Rad (1 mentions)
Anti gapdh, by Abbkine (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
Nunc maxisorp flat bottom 96 well plate, by Thermo Fisher Scientific (1 mentions)
Pierce tmb substrate kit, by Thermo Fisher Scientific (1 mentions)
17 protocols using pierce high sensitivity streptavidin hrp
1
Determining ErbB2 Aptamer Binding Affinity
2
IgE Binding Assay for rCan f 6 Allergen
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rCan f 6 and mutants were immobilised onto ELISA plates (IWAKI, Tokyo, Japan) overnight at 4 °C. After washing with PBS, the plate wells were blocked with 3% skim milk in PBS for 1 h at room temperature. Sera from 38 dog-allergic patients and 6 non-dog-allergic donors were diluted 1:250 with PBS, added to the wells (100 µL/well), and incubated for 1 h at 37 °C. Subsequently, biotin-labelled goat anti-human IgE antibody (0.5 mg/mL; Milford, MA, USA) diluted 1:5,000 with PBS was added to the wells (100 µL/well) and incubated for 1 h at 37 °C. Next, Pierce® High Sensitivity Streptavidin-HRP (1.1 mg/mL; Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:10,000 times with PBS was added (100 µL/well). For detection of allergen-IgE complexes, TMB Substrate Reagent (BD Biosciences, Bedford, MA, USA) was added (100 µL/well) and incubated for 15 min at room temperature. To stop the reaction, 1 N H2SO4 was used (100 µL/well). Absorbance at 450 nm was measured using the BioTekTM EonTM Microplate Spectrophotometer (BioTek Instruments Inc, Winooski, VT, USA).
3
SARS-CoV-2 Neutralizing Antibody Assay
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We used a validated in-house ELISA-based assay to estimate the degree of inhibition by the ACE-2 host receptor and RBD interaction as a proxy for neutralising capacity, as described elsewhere.15 (link),21 (link) This pseudo-neutralising assay correlates well with the gold standard plaque reduction neutralisation test (r = 0.9231).21 (link) In brief, recombinant ACE-2 ectodomain was coated onto Nunc Maxisorp 96-well plates in PBS overnight. For 1 h, a solution of patient serum, Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific), and biotinylated recombinant RBD was incubated in non-binding 96-well plates. The mixtures of biotinylated RBD/Streptavidin-HRP and patient serum were transferred to the ACE-2 ectodomain-coated wells for 35 min. Between each step, the wells were washed three times with PBS-T. Plates were developed for 20 min. The threshold for assay positivity was set to 25% inhibition in 10% diluted serum based on a receiver operating characteristic (ROC) curve analysis to estimate the optimal cut-off between naturally infected convalescent sera and sera from individuals obtained before 2020.8 (link),16 (link)
4
RNA Blotting and Visualization Procedure
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Hybond N + membranes (GE) were pre-incubated in 10× SSC. Precipitated biotinylated total RNA was dissolved in 2 μl of RNase free water. RNA was loaded onto the Hybond membrane and crosslinked using 254 nm ultraviolet light. The membrane was incubated with blocking solution (120 mM NaCl, 16 mM Na2HPO4, 8 mM NaH2PO4, 170 mM SDS) for 30 min. To the membrane was added 1 μl Pierce high sensitivity streptavidin-HRP(ThermoFisher) in blocking solution. The membrane was washed twice with wash buffer A (1:10 blocking solution) for 30 min, and twice with wash buffer B (100 mM Tris pH 9.5, 100 mM NaCl, 20 mM MgCl2) for 5 min. Membrane was incubated with Pierce western blotting substrate and visualized on the ChemiDoc (Biorad) under chemiluminesence hi sensitivity. To visualize RNA, the membrane was stained with a methylene blue solution (0.2% w/v methylene blue, 0.4 M sodium acetate).
5
Western Blot Analysis of Protein Expression
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After protein concentration was measured using the Lowry method, equivalent amounts of protein were prepared and separated on a 7.5% polyacrylamide-sodium dodecyl sulfate gel and transferred to an Immun-Blot® polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were washed three times with Tris-buffered saline (TBS; 0.02 M Tris and 0.5 M NaCl; Bio-Rad Laboratories, Inc.) containing 0.1% (v/v) Tween-20 (Bio-Rad Laboratories, Inc.). The membranes were successively incubated, first with blocking buffer containing TBS, 0.1% (v/v) Tween-20%, and 3% (w/v) albumin (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature. The next incubation was conducted with the primary antibodies anti-PKCα Clone M4 (EMD Millipore Corp., Billerica, MA, USA) and anti-GFP (Clontech Laboratories, Inc.) at a dilution of 1:10,000 for 1 h at room temperature. After washing twice with TBS containing 0.1% (v/v) Tween-20, a third incubation was performed with the secondary antibody anti-mouse Immunoglobulin G (IgG) biotin (eBioscience, Inc., San Diego, CA, USA) at a dilution of 1:10,000 for 1 h at room temperature. Finally, the membranes were probed with Pierce® High-Sensitivity Streptavidin-HRP (Thermo Fisher Scientific) at a dilution of 1:1000 for 1 h at room temperature.
6
Western Blot Analysis of RBCEV Proteins
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Antibody‐conjugated RBCEVs were lysed with RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (Biotool) for 15 min on ice. Total cell lysates were extracted by incubating with RIPA buffer supplemented with protease inhibitors for 30 min on ice. A total of 100 μg protein from RBCEVs and 30 μg protein from cell lysates were loaded in 10% polyacrylamide gels along with a protein ladder (Precision Plus Protein™ Kaleidoscope, Bio‐Rad), followed by a transfer to Immobilon‐P polyvinylidene difluoride membrane (Merck Millipore). The membrane was blocked with 5% milk in 1× Tris‐buffered saline containing 0.1% Tween‐20 (TBS‐T) at room temperature for 2 h followed by an incubation with primary antibodies: rabbit anti‐FLT3 (CST), mouse anti‐GAPDH (A01020, Abbkine, USA), Pierce™ High Sensitivity Streptavidin‐HRP (Thermo Fisher Scientific) overnight at 4°C. We washed the blots 3 times with TBS‐T, and then incubated them with HRP‐conjugated anti‐mouse and anti‐rabbit secondary antibodies (Santa Cruz, USA) for 1 h at room temperature. The blots were imaged using the Bio‐Rad Chemidoc gel documentation system.
7
Biotin-Labeled Aptamer Binding Assay
8
Quantification of Clostridium difficile Toxin B
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Toxin B concentrations from cecal contents were measured as previously described by Zarandi et al. 2017 and Girinathan et al., 2021 with the following adjustments [26 (link), 35 (link)]. Microtiter plates were coated with 5 μg/ml of the capture antibody (MAB13508, Abnova, Heidelberg, Germany). Plates were washed three times with PBS-Tween20 and blocked at RT for 2 h with SuperBlock (Thermo Scientific, Waltham, MA). Cecal contents were diluted with 1X PBS, vortexed, and centrifuged at 17,000 x g for 1 min. The sample supernatants and toxoid B standard controls (32-0 ng/ml) (BMLG1550050, Enzo Life Sciences, Southold, NY) were tested in triplicate. Plates were incubated with a biotinylated detection antibody (ab252712, Abcam, Cambridge, UK) and Pierce High-Sensitivity Streptavidin-HRP (Thermo Scientific, Waltham,MA). Plates were washed with PBS-Tween20 between incubations. TMB substrate (Thermo-Fisher, Waltham, MA) was used for signal detection at 450 nm with a BioTek Synergy H1 plate reader (Biotek Instruments Inc., Winooski, VT). Values were interpolated in GraphPad Prism 9 (GraphPad, San Diego, CA) to calculate toxin B concentrations. Significant differences between treatment groups were evaluated by non-parametric Mann-Whitney test with differences declared at a p-value ≤0.05.
9
Quantification of RGP-specific Immunoglobulins
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Serum total IgE, RGP‐specific‐IgE, and ‐IgG4 levels were measured by ImmunoCAP. Serum RGP‐specific IgG2 antibodies were measured by in‐house ELISA, as described previously.44 Briefly, ELISA plate wells were coated with an aqueous RGP extract (Stallergenes Greer), blocked with 2% bovine serum albumin in PBS (Sigma‐Aldrich), and incubated with serial dilutions of serum samples. Separate wells were coated with serial dilutions of purified human IgG2 (Sigma‐Aldrich, #I5404) to generate a standard curve for quantification of IgG2 in serum samples. Bound IgG2 was detected using biotinylated anti‐hIgG2 (clone HP6002; Thermo Scientific) followed by Pierce High Sensitivity Streptavidin‐HRP (Thermo Scientific). ELISA was developed using TMB (Thermo Scientific), and the reaction stopped with 1 mol/L HCl. Absorbance (OD 450 nm) was measured using a FLUOstar Optima plate reader (BMG Labtech).
10
Transglutaminase 2 Activity Assay
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ELISA wells were coated O/N at 4 °C for 16 h with 120 µL of 15 µg/mL N,N′-dimethyl casein (DMC) in PBS. After one wash with PBS, 100 µL of reaction mix (100 mM Tris-HCl, pH 8.0, 0.2 mM EZ-Link™ Pentylamine-Biotin from Thermo Scientific cat#21345, 10 mM DTT, 5 mM CaCl2) were added according to the test: (A) with increasing amounts of recombinant TG2; (B) with 50 ng of recombinant TG2 and increasing concentrations of CaCl2; (C) with 50 ng of recombinant TG2 in the presence or in the absence of 100 µM guanosine 5′- triphosphate (GTP); (D) with 50 ng of recombinant TG2 in the presence or in the absence of hTG2-specific peptide inhibitors 1-155, R281 or ZDON (Sigma-Aldrich, cat#616467) at three different concentrations (25 mM, 2.5 mM and 0.25 mM). Samples were added in each well and the plate was incubated for 1 h at 37 °C. Following three washes with PBS-Tween 20 0.1% and three with PBS, the plate was incubated for 1 h at 37 °C with 100 µL per well of Pierce™ High Sensitivity Streptavidin-HRP (Thermo Scientific, cat#21134) diluted in a solution of 1% BSA in PBS in volumetric ratio 1:200. After washes, the colorimetric reaction was developed by adding 100 µL per well of TMB and stopped after 5 min by adding 50 µL per well of 2.5 mM H2SO4. The absorbance was measured at 450 nm using a microplate reader.
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