SARS-CoV-2 mouse/rabbit Fc-tagged RBDs (final concentration 20 ng/ml) were incubated with serially diluted recombinant mAbs (from 25 µg/ml) and incubated for 1 h 37°C. The complex RBD:mAbs was then added to a pre-coated hACE2 (2 µg/ml in PBS) 96-well plate MaxiSorp (Nunc) and incubated 1 hour at room temperature. Subsequently, the plates were washed, and a goat anti-mouse/rabbit IgG (Southern Biotech) coupled to alkaline phosphatase (Jackson Immunoresearch) added to detect mouse Fc-tagged RBDs binding. After further washing, the substrate (p-NPP, Sigma) was added, and plates read at 405 nm using a microplate reader (Biotek). The percentage of inhibition was calculated as follow: (1-((OD sample-OD neg ctr)/(OD pos. ctr-OD neg. ctr))*100
Alkaline phosphatase
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters in an alkaline environment. It is commonly used as a reporter molecule in various immunological and molecular biology techniques.
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Lab products found in correlation
Goat anti mouse rabbit igg, by Southern Biotech (2 mentions)
Maxisorp, by Thermo Fisher Scientific (2 mentions)
A microplate reader, by Agilent Technologies (1 mentions)
Goat anti human, by Jackson ImmunoResearch (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Nuclear extraction kit, by Merck Group (1 mentions)
Timp 2, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
β actin antibody, by Abcam (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Fla 5000, by Fujifilm (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Attophos, by Promega (1 mentions)
Diaminobenzidine, by Vector Laboratories (1 mentions)
Vector blue, by Vector Laboratories (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Goat anti mouse horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
14 protocols using alkaline phosphatase
1
SARS-CoV-2 RBD Binding Inhibition Assay
2
Protein Characterization of Recombinant DsRed
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The TSP concentration was determined using a microtiter plate version of the Bradford method (Buyel and Fischer, 2014 (link)), and the protein composition was analyzed by lithium dodecylsulfate (LDS) polyacrylamide gel electrophoresis (PAGE) followed by gel staining with Coomassie Brilliant Blue (Menzel et al., 2016 (link)). DFE was quantified by fluorescence spectroscopy against DsRed standards (Buyel and Fischer, 2012 (link)). Briefly, DsRed fluorescence in the clarified extracts was measured using a Synergy HT microplate reader (BioTek Instruments, Winooski, Vermont, USA) fitted with 530/25 (excitation) and 590/35 (emission) nm filter sets. A standard curve was prepared with DsRed dilutions in the range 0–225 mg L−1, and the protein accumulation level per gram wet biomass was calculated as described elsewhere (Gengenbach et al., 2018 (link)). The presence of the C-terminal 2F5-epitope (ELDKWA) and His6 tag was confirmed by immunoblotting using an in-house preparation of the human monoclonal antibody 2F5 (Rühl et al., 2018 (link)) and a monoclonal rabbit anti-His6 antibody (BioVision, Milpitas, California, USA), respectively. These primary antibodies were detected using polyclonal goat-anti-human and goat-anti-rabbit immunoglobulin secondary antibodies, respectively, each conjugated to alkaline phosphatase (Jackson ImmunoResearch, Cambridge, UK).
3
Nuclear Protein Extraction and Western Blot
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Nuclear extraction kit (Sigma, St. Louis, MO, USA) was used to separate nuclear and cytosolic protein. Fifty micrograms of protein was loaded on SDS–PAGE, and then transferred to nitrocellulose sheets (NEN Life Science Products, Inc., Boston, MA, USA). After blocking, the blots were incubated with Nrf2, GPx, or b-actin antibody in 5% non-fat skimmed milk. After washed, the blots were incubated with secondary antibodies conjugated with alkaline phosphatase (Jackson Immuno Research Laboratories, Inc., Philadelphia, PA, USA). Immunoblots were developed using bromochloroindolyl phosphate/nitroblue tetrazolium solution (Kirkegaard and Perry Laboratories, Inc., Baltimore, MD, USA) (28 (link)).
4
Quantitative Western Blot Analysis
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A 50-microgram protein was loaded on SDS–PAGE, and then transferred to nitrocellulose sheets (NEN Life Science Products, Inc., Boston, Massachusetts). After blocking, the blots were incubated with MMP-2, MMP-9, TIMP-2, or β-actin antibody (Abcam, Cambridge, United Kingdom) (dilution 1:1000) in 5% non-fat skimmed milk (using β-actin as a loading control). After the blots were washed, they were incubated with secondary antibodies conjugated with alkaline phosphatase (dilution 1:3000) (Jackson ImmunoResearch Laboratories Inc., Philadelphia, Pennsylvania). Immunoblots were developed using bromochloroindolyl phosphate/nitroblue tetrazolium solution (Kirkegaard and Perry Laboratories Inc., Baltimore, Maryland).
5
Hormonal Regulation of Steroidogenesis
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Human FSH (hFSH) Human LH (hLH) and Human Chorionic Gonadotropin (hCG) were kindly provided by the NIH and Dr. Parlow. Deglycosilated hCG was enzymatically prepared as previously described [15 (link)]. Mouse monoclonal anti-diphospho ERK (anti-active ERK/MAPK) antibodies (DP-ERK Ab), and anti-general ERK antibody were obtained from Sigma, Israel (Rehovot, Israel). Anti-C-terminal ERK1 antibody (C16) was purchased from Santa Cruz. Polyclonal antibodies to human StAR were raised in rabbit [16 (link)]. Alkaline phosphatase, horseradish peroxidase and flourescein conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories Inc. (West Grove, Pennsylvania). PD98059 and U0126 were purchased from Calbiochem (San Diego). H89, Forskolin, 8-Br-cAMP were obtained from Sigma (St. Louis).
6
SARS-CoV-2 RBD-ACE2 Binding Inhibition Assay
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SARS-CoV and SARS-CoV-2 mouse/rabbit Fc-tagged RBDs (final concentration 20 ng/ml) were incubated with serially diluted recombinant mAbs (from 25 μg/ml) and incubated for 1 h 37°C. The complex RBD:mAbs was then added to a pre-coated hACE2 (2 μg/ml in PBS) 96-well plate MaxiSorp (Nunc) and incubated 1 hour at room temperature. Subsequently, the plates were washed and a goat anti-mouse/rabbit IgG (Southern Biotech) coupled to alkaline phosphatase (Jackson Immunoresearch) added to detect mouse Fc-tagged RBDs binding. After further washing, the substrate (p-NPP, Sigma) was added, and plates read at 405 nm using a microplate reader (Biotek). The percentage of inhibition was calculated as follow: (1-((OD sample-OD neg ctr)/(OD pos. ctr-OD neg. ctr))*100
7
Mapping Porcine Parvovirus Epitopes
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The peptides were synthesized based on the amino acid sequence of VP1 from PPV NADL-2 strain (GenBank, accession no. NC 001718) with 12 overlapping peptides and an offset of four peptides. The membrane used for spot synthesis was prepared with Fmoc (9-flurorenylmethoxycarbonyl) chemistry [23 , 24 (link)] on a PEG-derived cellulose membrane with the addition of a C-terminal anchor of β-Ala residues and hexadecapeptides at the N-terminal. All serum samples from D0, D28, D58 and D72 (representing, respectively, the basal serum condition prior to any intervention, the antibody response against the vaccine, the challenge with the reference strain and 21 days after the challenge) were screened for antibodies. An anti-swine IgG-Fc secondary antibody conjugated with alkaline phosphatase (Jackson ImmunoResearch Europe Ltd.) was used to assess epitope recognition in the peptide arrays and downstream procedures were done as previously described [24 (link)]. The membrane was subsequently scanned at 1200 d.p.i. with a Print Scan Copier (HP Photosmart) and the software package “Totallab Quant-Array Analysis” was used to quantify the spots based on the intensity of the reaction relative to the background intensity. Signal below the arbitrary threshold of 0.2 were considered negative.
8
Protein Identification by Western Blotting
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Protein identity was confirmed by western blotting using an in-house anti-PapMV CP polyclonal antibody or an anti-SARS-CoV-2 Spike Subunit 1 polyclonal antibody (Thermo Fisher, USA). An anti-rabbit antibody coupled with alkaline phosphatase (Jackson Immunoresearch, USA,) was used as a secondary antibody. Bands were coloured using step 1 nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a revealing reagent (ThermoFisher Scientific, Waltham, MA, USA).
9
Myc and Munc18-1 Protein Detection
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Samples were run on a 9% SDS-PAGE gel and transferred to Immuno-Blot PVDF Membrane (bio-rad). Blots were stained for myc (Genetex) and Munc18-1 (cell signalling). Secondary antibodies were conjugated with Alkaline Phosphatase (Jackson lab) and Attophos (Promega) was used as substrate. The blots were imaged with the Image Reader FLA-5000 (Fuji)
10
Immunohistochemical Analysis of Cardiac Allografts
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Cardiac allografts in untreated mice and mice exposed to the odor of TJ-23 were removed 30 days after transplantation and studied immunohistochemically with use of double immunostaining. Fresh 4-μm-thick graft cryosections were fixed in ice-cold acetone and preincubated in Block Ace (Dainippon Pharmaceutical Co., Ltd, Tokyo, Japan). Samples were incubated with anti-Foxp3 (kindly provided by Professor Kenjiro Matsuno, Dokkyo Medical University, Tochigi, Japan) polyclonal antibody; incubated with alkaline phosphatase (ALP)-conjugated anti-rabbit Ig (712-055-152; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for anti-Foxp3; and developed blue with Vector Blue (Vector Laboratories, Burlingame, CA). Cryosections were then incubated with rabbit anti-mouse type IV collagen polyclonal antibody (LB1403; Cosmo Bio, Tokyo) and peroxidase-conjugated anti-rabbit Ig (55693; Mitsubishi Chemical, Tokyo) and then developed brown with diaminobenzidine (Vector Laboratories).
Cardiac allografts in untreated mice and mice exposed to the odor of TJ-23 were removed 30 days after transplantation and studied histologically. Frozen sections (4-μm thick) were cut, mounted on silane-coated slides, and stained with hematoxylin-eosin.
Cardiac allografts in untreated mice and mice exposed to the odor of TJ-23 were removed 30 days after transplantation and studied histologically. Frozen sections (4-μm thick) were cut, mounted on silane-coated slides, and stained with hematoxylin-eosin.
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