The upper systemic three or four leaflets were collected from independent plants, frozen in liquid nitrogen, and suspended in urea SDS buffer (Levitzky et al., 2019 (link)). The proteins were extracted as in Levitzky et al. (2019 (link)) and separated on 12% SDS‐PAGE. The SDS‐PAGE gels were transferred onto nitrocellulose membranes by a semidry transfer blot (Bio‐Rad). Ponceau S staining was performed to visualize blotted proteins, and the RuBisCO small subunit (RbcS) band was used as a loading control. Phosphate‐buffered saline (PBS)‐washed membranes were blocked with PBS containing 4% nonfat milk, then incubated with anti‐CP polyclonal antibodies of ToBRFV, ToMV, or TMV, labelled with a secondary antirabbit antibody with horseradish peroxidase, and developed with EZ‐Link plus activated peroxidase kit (Thermo Fisher Scientific). Signals were captured in an Alliance Q9 Advanced imaging system (UVITEC). Image processing and quantification were performed with ImageJ (National Institutes of Health).
Ez link plus activated peroxidase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EZ-Link Plus Activated Peroxidase Kit is a laboratory product designed for the covalent conjugation of horseradish peroxidase (HRP) to proteins or other macromolecules. The kit contains pre-activated HRP and reaction buffers to facilitate the coupling process.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (2 mentions)
Hitrap, by Cytiva (2 mentions)
Alliance q9 advanced imaging system, by Uvitec (1 mentions)
Ez link sulfo nhs lc biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Skim milk, by Merck Group (1 mentions)
Tmb liquid substrate system for elisa, by Merck Group (1 mentions)
Enzymatic colorimetric assay, by Roche (1 mentions)
Xt 2000iv, by Sysmex (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Keyhole limpet hemocyanin (klh), by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Hitrap protein g hp kit, by GE Healthcare (1 mentions)
Enrofloxacin, by Merck Group (1 mentions)
Pefloxacin, by Merck Group (1 mentions)
21 protocols using ez link plus activated peroxidase kit
1
Quantitative Immunoblot Analysis of Viral Coat Proteins
2
Elephant IgM Antigen Identification
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Kyte-Doolittle Hyrophobicity and Hopp-Woods Hydrophilicity analysis of the predicted Asian elephant constant region of IgM was used to identify 3 antigenic peptides (Fig. 1 ). The peptides were synthesized and conjugated with KLH. Two rabbits each were inoculated with each peptide. Rabbits were inoculated and then boosted at days 14, 42, and 56. The rabbits were bled at day 72 and following confirmation that they had high titers towards the peptide were terminally bled at day 103. Approximately 50ml of each peptide specific serum was affinity purified using the same peptide used for immunization and then conjugated with HRP using the EZ-Link plus activated peroxidase kit (Thermo Scientific, Rockford, IL).
3
Purification and Conjugation of Monoclonal Antibody LM28
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LM28 was purified using the euglobulin precipitation protocol which involved hybridoma cell culture supernatant (250 mL) being dialysed for 3 days at 4 °C with 2 mM sodium phosphate buffer pH 6.0. The precipitated IgM was then centrifuged at 4 °C, 4000g for 10 min. The pellet was re-suspended and rinsed twice with cold 2 mM sodium phosphate buffer pH 6.0. Finally, the pellet was re-suspended in 10 mL of 1X PBS, centrifuged at 2000g for 10 min at RT. The supernatant was collected and aliquoted for storage. The efficiency of the purification was checked via SDS-PAGE, and the activity of the purified antibody was checked via immuno-dot assay on oat spelt glucuronoarabinoxylan (GAX). The concentration of the purified LM28 antibody was estimated by absorbance reading at 280 nm. The coupling of LM28 to HRP was performed using the EZ-link™ Plus Activated Peroxidase kit (Thermo Scientific) following manufacturer instructions.
4
Affinity Purification and Labeling of Anti-PTH Antibody
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pro[P1]PTH(−6 to +3) was conjugated to SulfoLink coupling gel via the C-terminal cysteine (PIERCE) for affinity purification of the rabbit antibodies, which were then HRP-labeled using EZ-Link Plus Activated Peroxidase Kit (Thermo Fisher Scientific), following the manufacturer’s protocol. The anti-pro[P1]PTH(−6 to +3) antibody was also conjugated to biotin using EZ-LinkTM Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific), following the manufacturer’s protocol, except using 40-fold instead of 20-fold excess of biotin. After biotin coupling, 15% of rabbit serum were added to the biotinylated antibody [anti-pro[P1]PTH(−6 to +3)Biotin].
5
Proximity Labeling of CD147 Interactome
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Cleavable tyramide-biotin was prepared by mixing 5 mg of EZ-Link™-biotin (Thermo Scientific) with 1.55 mg of tyramide hydrochloride (Sigma–Aldrich) in 100 µL of dimethyl sulfoxide in 2 mL of PBS. The mixture was incubated overnight at room temperature in the dark. After filtration, the tyramide-biotin was stored at − 20 °C until use. HRP was conjugated to anti-CD147 and huIgG using the EZ-Link™ Plus Activated Peroxidase kit (Thermo Scientific) according to the manufacturer’s instructions. For proximity labeling, cells (2 × 107) were washed with ice-cold PBS and incubated with anti-CD147-HRP Ab or huIgG-HRP (negative control) for 2 h at 4 °C. Following incubation, the cells were washed with ice-cold PBS and biotinylated for 15 min with tyramide-labeled buffer. After removal of unreacted reagents, the cells were lysed using RIPA lysis buffer (Thermo Scientific) via sonication on ice. Protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific). Biotin-labeled CD147 proximal proteins by anti-CD147-HRP were isolated using streptavidin magnetic beads (Thermo Scientific) according to the manufacturer’s instructions.
6
Competitive ELISA for Antigen Detection
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The competitive ELISA was performed as previously described with slight modifications.28 (link) Briefly, polystyrene microtiter plates were coated with 400 ng polyclonal IgG obtained from rabbits immunized by polypeptides in pH 9.6 carbonate-bicarbonate buffer overnight at 4°C. After three washes with phosphate buffer saline with 0.5% Tween 20, 300 μL of 5% skim milk were used to block remaining protein-binding sites. 50 μL diluted serum samples (1:2) were added to the wells, and 50μL proper diluted polypeptide coupled with horseradish peroxidase (HRP) using EZ-Link Plus Activated Peroxidase Kit (Thermo Scientific, Rockford, IL, USA) were added as quickly as possible. Then mix thoroughly, and the plates were incubated for 2 h at room temperature. After three washes, TMB substrate solution were added and the reactions were incubated for 30 min in dark. Then, 50 μL of 2 M H2SO4 was added to stop the reactions and optical density (OD) values were read at 450nm by a microplate reader. A standard curve was prepared meantime by plotting standard peptides (Genscript, Nanjing, China) concentration on x-axis versus OD values on the y-axis to determine the antigen concentrations of unknown tested samples. All tests were performed in triplicate on each plate.
7
Sandwich ELISA for mAb Specificity
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Sandwich ELISA was performed to determine the specificity of the selected mAbs. For this test, 1G10 mAb was conjugated with HRP using EZ-Link Plus Activated Peroxidase Kit (Thermo Scientific Inc.). Each well of a microtiter plate was coated with 0.1 μg of the 2CF5 mAb with overnight incubation. After 1 day, the plate was washed with PBST and blocked with 4% BSA in PBST for 2 hr, followed by a wash step and addition of a serial dilution of analytes (PvLDH antigen, P. falciparum 3D7, and P. vivax-infected patient blood) to the wells. After 1 hr incubation, the plate was washed, and 1G10 mAb conjugated HRP was added at 0.1 μg per well. An hour later, color was developed using TMB substrate and stopped by 0.18 M H2SO4. Absorbance at 450 nm was then read on a microplate reader.
8
Flag-Ism1 Protein Conjugation with HRP
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Flag-Ism1 protein was conjugated with HRP using the EZ-Link Plus Activated Peroxidase kit (#31489, Thermo Scientific) according to the protocol provided by the manufacturer. Briefly, Flag-Ism1 was conjugated with activated peroxidase (1:1) in Carbonate-Bicarbonate buffer (pH 9.4). After removing the Non-conjugated HRP, proteins were washed with RIPA buffer at least five times and eluted with 1 mg/ml Flag-peptide by shaking at 4 °C for 4–6 h, followed by concentration with the Amicon Filters Centrifuge (30 MWCO) to remove residual Flag peptide. Purified HRP-conjugated-Flag-Ism1 was checked by Coomassie Blue staining.
9
Antibody Binding Affinity to SARS-CoV-2 RBD
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Serial dilutions of each antibody (plant-made CA1 and CB6, and hybridoma-made 3C4 and 11D7) were coated overnight at 4 °C in 96-well plates. The plates were then incubated with recombinant WA1/2020 RBD at 2 µg/mL for 1 h at 37 °C. After washing with 1× PBST, the plates were further incubated with either CR3022 or CB6, both conjugated to HRP with the use of the EZ-Link Plus Activated Peroxidase Kit (Thermo Scientific). The plates were then developed with a KPL TMB substrate in conjunction with a 1M H2SO4 stop solution. Absorbance data were plotted with GraphPad Prism using nonlinear regression.
10
Development and Characterization of CCPP Blocking ELISA
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In a recently completed study [18 ], monoclonal antibodies (mAbs) against Mccp in the CCPP vaccine were generated and characterized at AU-PANVAC Laboratories (Debre-Zeit, Ethiopia). One of these mAbs (Mccp25) was used to develop the CCPP blocking-ELISA. This mAb was predominantly expressed and isotyped to belong to IgG2a, a subtype of immunoglobulin G. This Mccp-25 mAb was conjugated with HRP (Horse Redish Peroxodase) using the established method described in the manufacturer’s instructions for the EZ-Link Plus Activated Peroxidase kit l (PIERCE Cat. 31487; Thermo Scientific, Rockford, IL, USA). The conjugated mAb (Mccp25-HRP) was tested with CCPP b-ELISA and was easily blocked by antibodies from serum samples from CCPP vaccinated goats. The optimal dilution of the Mccp25-HRP conjugate and of the serum samples used in the CCPP b-ELISA was determined by testing two-fold serial dilutions of known strong and negative serum samples.
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