Shrimp-, lobster-, crab-, walnut-, and pecan-specific IgE, IgG, IgG1, IgG2a were quantified by ELISA, as previously reported (69 (link)). Briefly, plates were coated with 20 µg/ml food extracts (for samples) or 20 µg/ml HSA-DNP (for standard curves). After blocking with 2% BSA in PBS-0.05% Tween, serum samples were diluted 1:100 for IgE, 1:5,000 for IgG, 1:20,000 for IgG1 and 1:1,000 for IgG2a. Standard curves of mouse IgE anti-DNP, IgG1 anti-DNP or IgG2a anti-DNP (Accurate Chemicals, Westbury, NY) were generated ranging from 0.002–2 µg/ml. For IgE plates, the following detection antibodies were used in succession: 0.5 µg/ml sheep IgG anti-mouse IgE (The Binding Site, Birmingham, UK), 0.5 µg/ml biotinylated donkey IgG anti-sheep IgG (Accurate Chemicals), and 0.5 µg/ml NeutrAvidin-HRP (Pierce Biotechnology, Rockford, IL). For IgG, IgG1, and IgG2a plates, HRP goat anti-mouse IgG (Invitrogen, Waltham, MA), anti-mouse IgG1-HRP (Southern Biotech, Birmingham, AL), or anti-mouse IgG2a-HRP (Southern Biotech) were used, respectively. All plates were developed with TMB (SeraCare, Milford, MA), stopped with 1% HCl (SeraCare), and read on a plate spectrophotometer (BioTek, Winooski, VT) at 450 nm. Antigen-specific IgE, IgG1, and IgG2a concentrations were calculated based on the standard curve and dilution factor. Antigen-specific IgG is presented as O.D. values.
Neutravidin hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States
NeutrAvidin-HRP is a conjugate of NeutrAvidin and Horseradish Peroxidase (HRP) enzyme. NeutrAvidin is a deglycosylated form of avidin, a protein that binds to biotin with high affinity. The HRP enzyme provides a colorimetric detection signal when combined with appropriate substrates.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by LGC (3 mentions)
Hydrochloric acid (hcl), by LGC (3 mentions)
Anti mouse igg1 hrp, by Southern Biotech (1 mentions)
Plate spectrophotometer, by Agilent Technologies (1 mentions)
Anti mouse igg2a hrp, by Southern Biotech (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Microplate spectrophotometer, by Agilent Technologies (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Snakeskin dialysis tubing 10 k mwco, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti acrosin, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg conjugated with hrp, by GE Healthcare (1 mentions)
D biotin, by Thermo Fisher Scientific (1 mentions)
Donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Flag f7425, by Merck Group (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
10 protocols using neutravidin hrp
1
Quantification of Food-Specific Antibodies
2
Quantification of Food-Specific IgE Levels
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Walnut-, pecan- or egg-specific IgE was quantified via ELISA as described
previously [11 (link)]. Briefly, 96-well plates
were coated with 20 µg/mL walnut, pecan, or egg extract (for samples) or HSA-DNP
(for standard curves) and blocked with 2% BSA in PBS-0.5% Tween. Serum samples
were diluted 1:100, and standard curves ranging from 0.002-2 µg/mL of IgE
anti-DNP (Accurate Chemicals, Westbury, NY) were generated via 1:2 serial
dilutions. The following antibodies were used in succession for detection: sheep
IgG anti-mouse IgE (0.5 µg/mL, The Binding Site, Birmingham, UK), biotinylated
donkey anti-sheep IgG (0.5 µg/mL, Accurate Chemicals), and NeutrAvidin-HRP (0.5
µg/mL, Pierce Biotechnology, Waltham, MA). Plates were developed using TMB
(SeraCare, Milford, MA), stopped using 1% HCl (SeraCare), and read at 450 nm
using a microplate spectrophotometer (BioTek Instruments, Winooski, VT).
previously [11 (link)]. Briefly, 96-well plates
were coated with 20 µg/mL walnut, pecan, or egg extract (for samples) or HSA-DNP
(for standard curves) and blocked with 2% BSA in PBS-0.5% Tween. Serum samples
were diluted 1:100, and standard curves ranging from 0.002-2 µg/mL of IgE
anti-DNP (Accurate Chemicals, Westbury, NY) were generated via 1:2 serial
dilutions. The following antibodies were used in succession for detection: sheep
IgG anti-mouse IgE (0.5 µg/mL, The Binding Site, Birmingham, UK), biotinylated
donkey anti-sheep IgG (0.5 µg/mL, Accurate Chemicals), and NeutrAvidin-HRP (0.5
µg/mL, Pierce Biotechnology, Waltham, MA). Plates were developed using TMB
(SeraCare, Milford, MA), stopped using 1% HCl (SeraCare), and read at 450 nm
using a microplate spectrophotometer (BioTek Instruments, Winooski, VT).
3
Quantification of Antigen-Specific Antibodies
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Antigen-specific IgE, IgG1 and IgG2a were quantified by ELISA as described previously (21 (link)). Briefly, 96-well plates were coated with 20 µg/ml peanut, walnut, milk, or egg extract for samples, or 20 µg/ml HSA-DNP (Millipore Sigma, St. Louis, MO) for standard curves, and blocked with 2% BSA in PBS-0.5% Tween. Samples were diluted 1:100 for IgE, 1:20,000 for IgG1, and 1:1,000 for IgG2a. Standard curves were prepared using mouse IgE anti-DNP, IgG1 anti-DNP or IgG2a anti-DNP (Accurate Chemicals, Westbury, NY) ranging from 0.002–2 µg/ml. The following antibodies were added to IgE plates in succession: 0.5 µg/ml sheep IgG anti-mouse IgE (The Binding Site, Birmingham, UK), 0.5 µg/ml biotinylated donkey anti-sheep IgG (Accurate Chemicals), and 0.5 µg/ml NeutrAvidin-HRP (Pierce Biotechnology, Rockford, IL). For IgG1 and IgG2a plates, HRP goat anti-mouse IgG1 (1:40,000, Southern Biotech, Birmingham, AL) or IgG2a (1:20,000, Southern Biotech) were added to plates. All plates were developed with TMB (SeraCare, Milford, MA), stopped with 1% HCl (SeraCare), and read at 450 nm using a spectrophotometer (BioTek Instruments, Winooski, VT).
4
Biotinylation of US28 protein interactors
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HEK293T cells were transfected with only 250 ng pcDEF3-US28 Bio ID2 or 250 ng pcDEF3-US28 Bio ID2 and 2 μg pcDEF3-nanobody-mVenus. During the same day of transfection, medium was replaced with medium containing a final concentration of 50 μM biotin. The next day, cells were lysed in native lysis buffer (25 mM Tris HCL pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% Glycerol, 1 mM NaF, 1 mM NaVO3, cOmplete protease inhibitor cocktail) for 10 min on ice. Cell debris was removed by centrifugation at 13,000 × g. Cell lysate was dialyzed overnight using Snakeskin Dialysis Tubing 10 K MWCO (Thermo Fisher Scientific) to remove the excess of biotin. Protein concentration of lysates was determined by Pierce BCA protein assay kit (Thermo Fisher Scientific) and same protein quantities were separated on a 10% SDS-PAGE gel under reducing conditions and transferred to 0.45 μm PVDF blotting membrane (GE healthcare, Chicago, IL, USA). Biotinylated proteins were detected using neutravidin-HRP (1:2000 in 5% BSA/TBS-T, A2664, Thermo Fisher Scientific). Blots were developed using Western Lightning Plus-ECL (Perkin-Elmer, Waltham, MA, USA) and visualized with Chemidoc (Bio-Rad).
5
Western Blot Analysis of Sperm Proteins
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Proteins from the fractions were extracted by boiling in sample buffer [27 (link)] and separated by SDS-PAGE. Transfer, blocking, and immunodetection of specific proteins were performed largely as previously described [24 (link)]. Dilutions used for the primary antiserum were 1:4,000 for anti-α-tubulin (EMD Millipore, Millerica, MA, USA) and 1:500 for anti-acrosin (Santa Cruz Biotechnology, Dallas, TX, USA), and for the biotin-binding probe was 1:100,000 for NeutrAvidin-HRP (Thermo Fischer Scientific). A 1:5,000 dilution was used for anti-mouse IgG conjugated with HRP (GE Healthcare Life Sciences, Pittsburg, PA, USA). Chemiluminescence was used to detect immunoreactivity.
6
Antibody Immunofluorescence and Western Blot
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Mouse monoclonal antibodies: acetylated Tubulin (T7451, IF: 1:10,000; Sigma-Aldrich), ARL13B (66739-1-Ig, IF: 1:300; Proteintech, 75-287, IF: 1:1,000; NeuroMab), and S-tag (MAC112, IF: 1:100, WB: 1:5,000; EMD Millipore). Rat monoclonal antibody: HA (7c9, WB: 1:1,000; ChromoTek). Rabbit polyclonal antibodies: EGFP (50403-2-AP, IF: 1:200, WB: 1:1,000; Proteintech), Flag (F7425, WB: 1:1,000; Sigma-Aldrich), Adenylyl cyclase III (C-20, IF: 1:100; Santa Cruz), B9D1 (NBP2-84489, WB: 1:1,000; Novus Biologicals) and HTR6 (31 (link)). Goat polyclonal antibodies: γ-Tubulin (sc-7396, IF: 1:200; Santa Cruz). Alexa Fluor (AF)–conjugated donkey secondary antibodies from Thermo Fisher Scientific (all used for IF at 1:10,000): AF488 anti-mouse IgG (A21202), AF488 anti-rabbit IgG (A21206), AF555 anti-mouse IgG (A31570), AF555 anti-rabbit IgG (A31572), AF594 anti-rabbit IgG (A21207), and AF647 anti-goat IgG (A21447). Also from Thermo Fisher Scientific were HRP-conjugated secondary antibodies (used for WB at 62 ng/ml): goat anti-rabbit IgG (A16104), goat anti-mouse IgG (A16072) and donkey anti-rat IgG (A18739). Biotinylated proteins were detected with Neutravidin-HRP (A2664, 1 μg/ml; Thermo Fisher Scientific). D-biotin was from Thermo Fisher Scientific (BP-232-1).
7
PGA1-Biotin Protein Detection by Western Blot
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Polyacrylamide gel electrophoresis was performed under denaturing conditions. Proteins were separated using 10% acrylamide gels. After SDS-PAGE, proteins were transferred to PVDF membrane (Merck, Darmstadt, Germany) and PGA1-biotin labeled proteins were incubated with NeutrAvidin-HRP (Thermo Scientific, Bonn, Germany) at a dilution of 1:10,000 in TBST including 3% non-fat dried milk powder. Chemoluminescence detection was done using ImmobilonTM HRP substrate (Millipore, Billerica, MA) according to the manufacturer`s protocol. After signal detection, western blots were stained with Coomassie to visualize protein loading. For size determination 5 μL PageRuler Plus Prestained Protein Ladder or PageRuler Prestained Protein Ladder (Fermentas, St. Leon-Rot, Germany) was used.
For experiments with [14C]-labeled OPDA a 12% Precise Protein Gel (Thermo scientific, Bonn, Germany) was used, stained by Coomassie G250 (Applichem, Darmstadt, Germany) and radioactivity was detected by using a Fujix BAS 2000 system (Fuji Photo Film, Tokyo, Japan)
For experiments with [14C]-labeled OPDA a 12% Precise Protein Gel (Thermo scientific, Bonn, Germany) was used, stained by Coomassie G250 (Applichem, Darmstadt, Germany) and radioactivity was detected by using a Fujix BAS 2000 system (Fuji Photo Film, Tokyo, Japan)
8
CD137L Binding Inhibition Assay
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ADG106 was tested for its ability to block the binding of the human CD137 ligand (CD137L)-Fc fusion protein (Sino Biological) to plate-bound recombinant CD137-Fc fusion protein (Sino Biological).
Nunc-Immuno MaxiSorp surface 96-well plates were coated with 0.1 mL/well of the 1 μg/mL CD137 solution overnight at 4°C. The CD137 ligand was biotinylated and purified using the EZ-Link sulfo-NHS-SS-biotin kit (Pierce) according to the manufacturer’s instructions.
The following day the CD137 coated wells were blocked with 0.3 mL of a blocking buffer (2% BSA in PBS) at room temperature for 1 h. ADG106 and a control human IgG4 antibody were diluted in the range from 500 μg/mL to 0.038 μg/mL in 2% BSA-PBS. Biotinylated CD137L was diluted to 4 μg/mL in 2% BSA-PBS.
Then, 50 μL of the antibody dilutions, along with 50 μL biotinylated CD137L, were added to the blocked CD137-coated wells and incubated at 37°C for 1 h. The plates were washed with 0.2 mL of wash buffer (0.05% Tween 20 in PBS).
Subsequently, 100 μL/well of the Neutravidin-HRP (Thermo fisher) diluted at 1:5000 in 2% BSA-PBS, was added to the washed plates and incubated at 37°C for 1 h. After further washing, 100 μL TMB substrate solution was added to each well and incubated at room temperature for 15 min. The reaction was halted with 100 μL 1N H2SO4, and the absorbance at 450 nm was measured using a BioTEK plate reader.
Nunc-Immuno MaxiSorp surface 96-well plates were coated with 0.1 mL/well of the 1 μg/mL CD137 solution overnight at 4°C. The CD137 ligand was biotinylated and purified using the EZ-Link sulfo-NHS-SS-biotin kit (Pierce) according to the manufacturer’s instructions.
The following day the CD137 coated wells were blocked with 0.3 mL of a blocking buffer (2% BSA in PBS) at room temperature for 1 h. ADG106 and a control human IgG4 antibody were diluted in the range from 500 μg/mL to 0.038 μg/mL in 2% BSA-PBS. Biotinylated CD137L was diluted to 4 μg/mL in 2% BSA-PBS.
Then, 50 μL of the antibody dilutions, along with 50 μL biotinylated CD137L, were added to the blocked CD137-coated wells and incubated at 37°C for 1 h. The plates were washed with 0.2 mL of wash buffer (0.05% Tween 20 in PBS).
Subsequently, 100 μL/well of the Neutravidin-HRP (Thermo fisher) diluted at 1:5000 in 2% BSA-PBS, was added to the washed plates and incubated at 37°C for 1 h. After further washing, 100 μL TMB substrate solution was added to each well and incubated at room temperature for 15 min. The reaction was halted with 100 μL 1N H2SO4, and the absorbance at 450 nm was measured using a BioTEK plate reader.
9
Quantifying Peptide Binding to Extracellular Vesicles
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The 96-well ELISA plates with 100 µL (20 mg/mL) of target EVs overnight at 4 °C. The wells were blocked (TBS with 1% BSA) followed by the addition of biotinylated peptides (0–160 mg/mL diluted in 1× TBS) and incubated overnight at 4 °C. Bound peptides were detected with NeutrAvidin-HRP (Thermo #31001, Waltham, MA, USA, 1:10,000 in TBS) for 2 h at room temperature with shaking. TMB color detection was performed as described above.
10
Mouse/Human IL1RAPL1 Binding Assay
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Mouse or human IL1RAPL1/Fc chimera (Sino Biological Inc.) were immobilized on 384 maxisorp plates (NUNC) at a concentration of 0.1 mg/well in 25 ml PBS overnight at 4 C. After blocking with 90 ml PBT-buffer (0.5% BSA, 0.1% Tween 20 in PBS) for 1 h and 4 C, a 1:1 dilution series of recombinant mouse or human His-Ub-IL-38-Avi fusion protein (0, 0.031, 0.063, 0.125, 0.25, 0.5 and 1 mM) were added to the wells and incubated for 2 h at 4 C in PBT buffer. After IL-38 binding, wells were washed 4 times with 90 ml PT buffer (0.1% Tween 20 in PBS). Subsequently, 25 mL of High Sensitivity NeutrAvidin-HRP (Thermo Fisher Scientific, diluted 1:2000 in PBT buffer) was added to the wells. After incubation for 1 h at 4 C, the plate was washed 4 times with PT buffer and 25 ml freshly prepared Tetramethylbenzidine (TMB) substrate (BD Biosciences) was added. The reaction was stopped with 25 mL 1.0 M H3PO4. Signals were read spectrophotometrically at 450 nm (Epoch, BioTek). Plotted values were corrected for background binding of mouse His-Ub-IL-38-Avi to BSA coated wells.
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