Primary fibroblasts were cultured from a skin biopsy sample from Pt1, Pt2, or a control subject in Dulbecco modified Eagle medium supplemented with 10% FBS, 2 mmol/L glutamine, 10 mmol/L HEPES, and 40 μg/mL gentamicin (Life Technologies, Courtaboeuf, France). Fibroblasts were transfected with either pMax-GFP (Lonza, Basel, Switzerland) vector or TrueORF gold vector coding for Myc-DDK-tagged ORF of human AIRE transcript variant AIRE-1 (OriGene Technologies, Rockville, Md) by using the jetPEI reagent (Polyplus Transfection, Illkirch, France). After 24 hours, fibroblasts were lysed in NP-40 lysis buffer (20 mmol/L Tris/HCl (pH 7.4], 150 mmol/L NaCl, 2 mmol/L EDTA, and 1% NP-40 (Sigma-Aldrich]) containing protease inhibitors for 30 minutes at 48C. Supernatants were collected after 10 minutes of centrifugation at 16,000g and 48C, and protein content was quantified with the mBCA quantification kit (Thermo Fisher Scientific Biosciences, Villebon sur Yvette, France). Protein extracts (50 μg) were analyzed by using Western blotting with anti-AIRE antibody (Abnova, Taipei, Taiwan), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:10,000, Sigma-Aldrich), and a BM Chemiluminescence Blotting Substrate Kit (Roche).
Anti rabbit igg antibody conjugated to horseradish peroxidase
Manufactured by Merck Group
Sourced in China
The anti-rabbit IgG antibody conjugated to horseradish peroxidase is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that binds to rabbit immunoglobulin G (IgG) and is coupled with the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Np 40, by Merck Group (1 mentions)
Jetpei reagent, by Polyplus Transfection (1 mentions)
Pmaxgfp, by Lonza (1 mentions)
Bm chemiluminescence blotting substrate kit, by Roche (1 mentions)
Sars cov 2 rbd polyclonal antibody, by Sino Biological (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Gradient sds page, by Bio-Rad (1 mentions)
4 protocols using anti rabbit igg antibody conjugated to horseradish peroxidase
1
Fibroblast Transfection and AIRE Expression
2
Protein-Protein Interaction Assay
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To evaluate the interaction between TcCaNA2 and TcCaNB, a Far-Western blotting was performed as described by Wu et al.[23] (link) using the recombinant proteins TcCaNB (tagged with GST) and TcCaNA2 (tagged with 6×His). BSA and the unrelated fusion protein 6×His-SUMO-CAT were used as negative controls. One microgram each of BSA, CAT and 6×His-SUMO-TcCaNA2 (target protein) were resolved in 10% SDS-PAGE and transferred onto PVDF membranes, which were then incubated with decreasing concentrations of guanidine-HCl (6, 3, 1, 0.1, and 0 M) to denature and renature the target protein. The membrane was then blocked with PBS containing 0.5% Tween 20 and 5% skim milk and incubated with 10 µg of the bait protein GST-TcCaNB. A rabbit polyclonal antibody directed to GST-TcCaNB and an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Sigma-Aldrich) were used to detect TcCaNA2-TcCaNB interaction. The immunocomplexes were revealed using diaminobenzidine (DAB) as substrate.
3
SARS-CoV-2 Spike Protein RBD Detection
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Cell lysates from 143B cells after 24‐h infection with SCV‐S or control vector were analyzed by immunoblotting using the SARS‐CoV‐2 RBD polyclonal antibody (catalog number 40592‐T62; Sino Biological, Beijing, China; 1:1000) and anti‐rabbit IgG antibody conjugated to horseradish peroxidase (catalog number A9044; Sigma Aldrich, St. Louis, MO, USA; 1:10 000) and imaged using ChemiDoc XRS (Bio‐Rad Laboratories, Hercules, CA, USA).
4
Anisakidae species identification by SDS-PAGE and Western blot
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Whole parasite extracts were analyzed by 4-20% gradient SDS-PAGE (BioRad, Hercules, California) under reducing conditions (Laemmli, 1970) , and gels were stained with colloidal Coomassie G-250 (PageBlue, Thermo Fisher Scientific, Rockford, Illinois). For WB analysis, parasite extracts from the 3 species were first separated by 4-20% gradient SDS-PAGE in duplicate and then transferred onto a 0.45-lm nitrocellulose membrane (BioRad, Mu¨nchen, Germany). After the blocking step, the membrane was cut; 3 strips loaded with different Anisakidae species extracts were incubated with rabbit anti-A. simplex hyperimmune serum diluted 1:400; another 3 strips were incubated with preimmune rabbit serum. The anti-rabbit IgG antibody conjugated to horseradish peroxidase (Sigma) was used as a secondary antibody. SDS-PAGE and WB profiles were analyzed with GelAnalyzer software (ver. 2010a; http://www.gelanalyzer.com/).
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