For PrP analysis in brain extracts, brain homogenates (3 different animals) prepared in PBS were either not digested or treated with different concentrations of PK (0 to 5 mg/ml; VWR, Ca) as indicated for one hour at 37°C. The reaction was terminated by adding 1X pefabloc proteinase inhibitor (VWR, Ca). Fifty μg of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to PVDF membranes (Millipore, Ca). PVDF membranes were probed using anti-PrP monoclonal antibodies followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma, Ca) and developed using ECL-plus detection (Amersham). Images were acquired on X-ray film (Super Rx; Fujifilm) or by using a digital imaging system (Alpha Innotech, FluoriChemQ). FluoChemQ software (Alpha Innotech) was used to quantify and determine the relative values of PrPres signals.
Horseradish peroxidase conjugated goat anti mouse igg antibody
Manufactured by Merck Group
Sourced in United States, Germany
Horseradish peroxidase-conjugated goat anti-mouse IgG antibody is a laboratory reagent used for the detection of mouse immunoglobulin G (IgG) in various immunoassays. The antibody is produced in goats and is labeled with the enzyme horseradish peroxidase, which can be used as a reporter molecule to generate a colorimetric or chemiluminescent signal when exposed to appropriate substrates.
Automatically generated - may contain errors
Lab products found in correlation
Digital science id image analysis software, by Kodak (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Coomassie plus protein assay kit, by Thermo Fisher Scientific (3 mentions)
Pefabloc proteinase inhibitor, by Avantor (2 mentions)
Super rx, by Fujifilm (2 mentions)
Anti vegf, by Santa Cruz Biotechnology (2 mentions)
Anti phosphoinositide 3 kinases pi3k, by Cell Signaling Technology (2 mentions)
Anti vegfr 2, by Merck Group (2 mentions)
Gtx629630, by GeneTex (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fluorichemq, by Bio-Techne (1 mentions)
Fluochemq software, by Bio-Techne (1 mentions)
Endosafe endochrome k kit, by Charles River Laboratories (1 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Phosphatidylcholine pc, by Avanti Polar Lipids (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phosphorylated iκbα, by Cell Signaling Technology (1 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
21 protocols using horseradish peroxidase conjugated goat anti mouse igg antibody
1
PrP Detection in Brain Extracts
2
Immunoassay Reagents and Materials
3
Measuring Signaling Pathways in Lung Tissue
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The protein extracts of lung were incubated with the primary antibody [anti-phosphoinositide 3-kinases (PI3K) (1:1000; Cell Signaling Technology); anti-nuclear factor kappa B (NFκB, p65) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.); anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) (1:10000; Abcam plc); anti-phosphorylated IκBα (1:1000; Cell Signaling Technology); anti-VEGF (1:1000; Santa Cruz Biotechnology); anti-VEGFR-1, -phosphorylated VEGFR-1 (1:1000; Abcam plc); anti-VEGFR-2 (1:500; Millipore Corporation); anti-phosphorylated VEGFR-2 (1:1000; Cell Signaling Technology); anti-Rho-associated kinase (RhoA) (1:1000; Cell Signaling Technology); anti-Akt (1:500, Cell Signaling Technology); anti-phosphorylated Akt (1:2000, Cell Signaling Technology); anti-extracellular signal-regulated kinase (ERK), -phosphorylated ERK (1:3000, Millipore Corporation)]. Then the blots were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG antibody, Sigma Chemical Co., St. Louis, MO, U.S.A.). With a computer assisted video densitometer and digitalized software (Kodak Digital ScienceTM ID Image Analysis Software, Eastman Kodak Co., Rochester, NY, U.S.A.), the blots were scanned, photographed then the signal intensity (integral volume) of the appropriate bands were analyzed.
4
Quantifying Allergen-Specific IgG in ELISA
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Nunc Maxisorp plates (Fisher Scientific, Schwerte, Germany) were coated overnight at room temperature with 250 ng/100 µl proteins (Δ29NCS_5x; Δ29NCS; Dau c 1; Pru av 1; Bet v 1; Gly m 4; Cor a 1), with 50 ng/100 µl Api g 1, and with 25 ng/100 µl Bet v 1 and Bet v 1_4x in phosphate-buffered saline (PBS). After blocking with PBS containing 2% BSA a dilution series of the murine monoclonal anti-Bet v 1 antibody BV16 [34] (link) was added for 1 h at room temperature in PBS containing 0.05% Tween 20 and 0.1% BSA. Allergen-specific IgG was detected with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (A3673, SigmaAldrich, Taufkirchen, Germany) diluted 1∶3000 in PBS containing 0.05% Tween 20 and 0.1% BSA as described for the IgE ELISA below.
5
Western Blot Analysis of Liver Proteins
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Liver tissues were frozen in liquid nitrogen and stored at −80 ℃ until required for Western blot analysis. Blots were incubated with the primary antibody (endothelial nitric oxide synthase (eNOS) (Cell Signaling Technology, Danvers, Massachusetts, USA. 32027S; 1:1000), inducible nitric oxide synthase (iNOS) (GeneTex International Corporation, Hsinchu, Taiwan, Gtx130246; 1:1000), LOX-1 (GeneTex GTX 59636; 1:3000), NFκB (Cell Signaling 8242S; 1:3000), IκBα (Cell Signaling 4814S; 1:3000), beta-actin (Genetex Gtx629630; 1:5000)); then, the blots were incubated for 90 min with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG antibody; Sigma Chemical Co., St. Louis, MO, USA). The specific proteins were detected by enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Merk Millipore Co., Billerica, MA, USA) and scanned with a computer-assisted video densitometer and digitalized system (BioSpectrum® 600 Imaging System, Ultra-Violet Products Ltd., Upland, CA, USA). Then, the signal intensity (integral volume) of the appropriate band was analyzed.
6
Detecting Autoantibody and Cytokine Levels
7
Chicken Fibroblast and HEK293T Cell Maintenance
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The chicken fibroblast cell line (Douglas Foster 1, DF-1) and human embryo kidney cell line (293T) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The DF-1 and 293T cells were cultured at 37 °C in a cell incubator under 5% CO2. The cell lines were regularly tested in-house for mycoplasma infection. RIPA buffer, streptomycin, penicillin, mouse anti-β-actin monoclonal antibody (mAb), horseradish-peroxidase-conjugated goat anti-mouse IgG antibody, and hemagglutinin (HA)-tagged and FLAG-tagged mAbs were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Chicken Interferon Alpha was purchased from Bio-Rad (Hercules, CA, USA). Rabbit anti-phospho-STAT1 (p-STAT; S727), anti-STAT1 (ab92506), anti-Mertk (ab300136) were purchased from Abcam (Cambridge, UK). SDS-PAGE sample loading buffer and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime (Nantong, China). Mut Express® II Fast Mutagenesis Kit V2 was purchased from Vazyme Biotect Co., Ltd. (Nanjing, China) for site-directed mutagenesis. TRIzol Reagent, FBS, DMEM, enhanced chemiluminescent reagent (ECL), Alexa-Fluor®−594- and Alexa-Fluor®−488-conjugated goat anti-mouse secondary antibodies were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). The mAb directed against the NDV nucleoprotein (NP) was prepared in our laboratory.
8
Western Blot Analysis of Liver Proteins
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Liver tissues were frozen in liquid nitrogen and stored at -80°C until Western blot analysis. Blots were incubated with the primary antibodies (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α
NPC1L1 [1:1000, GeneTex GTX30599; USA], beta-actin [1:5000, GeneTex Gtx629630; Irvine, CA, USA]), and then the blots were incubated with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG antibody; Sigma Chemical Co., St. Louis, MO). Specific proteins were detected by enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate; Merk Millipore Co., Billerica, MA) and scanned with a computer-assisted video densitometer and digitalization system (BioSpectrum® 600 Imaging System; Ultra-Violet Products Ltd., Upland, CA). Signal intensity (integral volume) of the appropriate band was then analyzed.
NPC1L1 [1:1000, GeneTex GTX30599; USA], beta-actin [1:5000, GeneTex Gtx629630; Irvine, CA, USA]), and then the blots were incubated with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG antibody; Sigma Chemical Co., St. Louis, MO). Specific proteins were detected by enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate; Merk Millipore Co., Billerica, MA) and scanned with a computer-assisted video densitometer and digitalization system (BioSpectrum® 600 Imaging System; Ultra-Violet Products Ltd., Upland, CA). Signal intensity (integral volume) of the appropriate band was then analyzed.
9
PrP Detection in Brain Extracts
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For PrP analysis in brain extracts of different animal models, brain homogenates prepared in PBS were digested with different concentrations of proteinase K (PK; Roche) for 1 hour at 37°C. The enzymatic reaction was terminated by the addition of 1X pefabloc proteinase inhibitor (VWR), then samples were denatured at 96°C for 10 min in 3X SDS (sodium dodecyl sulphate) sample buffer. For deglycosylation of PK-digested and denatured samples, PNGase F enzyme (Roche) was added to the samples according to the manufacturer’s instructions. Samples were resolved on 12% NuPAGE bis-tris gels (Life Technologies), and then electrophoretically transferred to PVDF membranes (Millipore, Ca). PVDF membranes were probed using anti-PrP monoclonal antibodies followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) and developed using ECL-plus detection (Amersham). Images were acquired on X-ray film (Super Rx; Fujifilm).
10
Recombinant human GAA Characterization
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