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Goat anti mouse igg

Manufactured by Southern Biotech
Sourced in United States, France

Goat anti-mouse IgG is a purified polyclonal antibody produced in goats and specifically binds to mouse immunoglobulin G (IgG). It is commonly used as a secondary antibody in immunoassays and other applications that require the detection of mouse primary antibodies.

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71 protocols using goat anti mouse igg

1

Quantifying Immunoglobulins in Plasma and Feces

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Blood and feces were sampled. Intracardiac blood was collected with a heparinized syringe and recovered plasma was kept at − 80 °C. Fecal proteins were extracted mechanically in complete antiprotease cocktail (Roche Diagnostic, Meylan, France) and frozen at − 80 °C. Plasma and fecal IgG and IgA concentrations were measured by ELISA.
Plates were coated overnight at + 4 °C with 5 μg/ml sheep anti-mouse IgA (Sigma-Aldrich) or goat anti-mouse IgG (Southern Biotech, France) in PBS. Plates were blocked with PBS-5% fetal calf serum (FCS) (Invitrogen) before incubation with diluted samples or purified IgA or IgG (Southern Biotech). Horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgA (Sigma-Aldrich) or goat anti-mouse IgG (Southern Biotech) were added, HRP was revealed using TMB (Becton Dickinson, France). Reaction was stopped adding H2SO4 2 N and plates were analysed using automatic Infinite M200 microplate reader. Measurements were performed in duplicate and detection threshold correspond to at least three blank values.
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2

Mouse Immunoglobulin ELISA Protocol

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Plates were coated with 5 µg/ml of sheep anti-mouse IgA (Sigma) or goat anti-mouse IgG (SouthernBiotech, Cliniscience, Nanterre, France), incubated with plasma, detected with 1.5 µg/ml HRP-conjugated goat anti-mouse IgA (Sigma) or goat anti-mouse IgG (SouthernBiotech). HRP was revealed using TMB and the reaction was stopped with H2SO4 before reading at 450 nm using Varioskan. microplate reader.
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3

Immunoglobulin and Anti-dsDNA Antibody Quantification

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Serum samples obtained from all mice groups at different age intervals were tested for the presence of total immunoglobulins and anti-dsDNA antibodies using ELISA assays. In the case of total immunoglobulin detection, microplates were coated with 2 µg/ml goat anti-mouse IgG (Southern Biotech) or IgM. Following blocking, serially diluted immunoglobulin (Ig) standards (IgG, Catalog# I5381, Sigma-Aldrich; mouse IgM, catalog# 010-0107, Rockland) or serum samples were added into the microplate. After a two-hour incubation at room temperature, the bound Igs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Catalog# 2040-04, Southern Biotech) or goat anti-mouse IgM (Catalog# 626822, Invitrogen). Subsequently, quantification was carried out by measuring the absorbance at 405 nm. For the detection of IgG anti-dsDNA antibodies, 100 μg/ml of mBSA in phosphate-buffered saline (PBS) was applied to pre-coat the plates at 37°C for thirty minutes prior to the incubation of dsDNA standards (Catalog# D1501, Sigma) with an initial concentration at 1250 ng/ml. Serum samples were diluted 1:50 in PBS and added to the plates for a two-hour incubation. Alkaline phosphatase-conjugated goat anti-mouse IgG was used as the detection antibody.
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4

Comprehensive Immunological Assay Protocol

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For flow cytometry, immunofluorescence, ELISA, and cell stimulation, antibodies to mouse CD3ε (145-2C11) and CD28 (37.51) were from BioXCell; antibodies to mouse CD4 (RM4-5), CD4 (GK1.5), CD25 (PC61.5), CD44 (IM7), CD62L (MEL-14), CD45.1 (A20), CD45.2 (104), B220 (RA3-6B2), ICOS (7E.17G9), PD-1 (J43), Ki-67 (SolA15), IFN-γ (XMG1.2), IL-2 (JES6-5H4), IgD (11-26c), Annexin V, streptavidin, and Fixable Viability Dye eFluor 450 were from eBioscience; antibodies to mouse CD4 (RM4-5), CXCR5 (2G8), Bcl-6 (K112-91), CD95 (Jo2), the T cell– and B cell–activation antigen (GL7), and streptavidin were from BD PharMingen; and antibodies to CD8 (53–6.7), CD150 (SLAM, TC15-12F12.2), CD138 (281–2), and streptavidin were from BioLegend. Biotin-conjugated goat antibody to rat IgG (112–065-167) was from Jackson ImmunoResearch. Antibody to HIF-1α (241812) was from R&D Systems. Biotinylated PNA (B-1075) was from Vector Laboratories. Goat anti-mouse IgG (1030–08) was from SouthernBiotech. For immunoprecipitation assay and immunoblot analysis, antibodies to Myc (sc-40 [9E10]) and VHL (sc-5575 [FL-181]) were from Santa Cruz Biotechnology; antibody to actin (A2228 [AC-74]) was from Sigma-Aldrich; antibody to m6A (61496) was from Active Motif; and polyclonal rabbit IgG (isotype control, ab171870) was from Abcam.
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5

Autoantibody Profiling in Mouse Models

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The levels of autoantibodies specific for selected linear peptides in the sera
of B6 and BXD2 mice were determined by ELISA using a NeutrAvidin High Binding
Capacity-coated 96-well plate (Thermo Scientific Pierce). Briefly, biotinylated peptides
were conjugated to the plate overnight at 4°C at a concentration of 30 µM
(all peptides were purchased from Sigma Aldrich). ELISAs were developed with either an
HRP-labeled goat anti-mouse IgG or a goat anti-mouse IgM Ab (Southern Biotechnology
Associates) and tetramethylbenzidine substrate (Sigma-Aldrich).
OD450–650 was measured on an Emax Microplate reader.
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6

Quantifying IgG-secreting Antibody-Forming Cells

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ELISpot plates (Millipore, Ref# MSIPS4W10; Multiscreen HTS) were coated with 0.01% poly-L lysine for 1h at RT, followed by coating with 100 μg/ ml calf thymus DNA (Sigma Aldrich) or 10 μg/ ml calf thymus nucleosomes (Arotec diagnostics) overnight at 4°C. Blocked with PBS + 5% FCS for 2–3h at RT. Freshly isolated splenocytes or BM cells were resuspended in freshly prepared warm 15% RPMI 1640 media + antibiotics + 1mM L-glutamine at 20 × 106 cells/ ml. 1×106 total cells were plated on the top wells and serially double diluted (1:2). ELISpot plates were then incubated for 18h at 37°C with 5% CO2. Washed with PBS + 1% FCS to remove the cells and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch) for 3–4h at 4°C. After washing, plates were developed using the Vector Blue AP Substrate Kit III (Vector Labs). Spots were captured and counted using ImmunoSpot S6 Analyzer (Cellular Technology Limited, Shaker Heights, OH). For analysis of total IgG-secreting AFCs, ELISPot plates were coated with goat anti-mouse IgG (SouthernBiotech) at 5μg/ml and blocked with PBS + 5% BSA. Plates were washed with PBS + 1% BSA and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch).
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7

Measuring Intestinal sIgA Levels by ELISA

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The sIgA levels in intestinal lavage were determined using a capture ELISA. The colon was washed with 5 mL of PBS1X, and the fluid was centrifuged at 1200 rpm and 4 °C for 10 min. The sIgA levels in the supernatant were measured using goat anti-mouse IgG, human ads-UNLB and goat anti-mouse IgA conjugated with horseradish peroxidase (HRP) antibodies (Southern Biotech, Birmingham, AL, USA) according to the manufacturer's instructions. The absorbance (492 nm) was measured using a POLARIS MA616—Marconi instrument (Piracicaba, Brazil).
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8

Quantification of Mouse Ig Isotypes

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For detection of antibodies in serum, plates were coated with rat-anti-mouse IgE (R35-72; BD) or goat-anti-mouse IgG (Southern Biotech) overnight at 4°C. Purified mouse IgE (BD) or unlabelled mouse IgG1, IgG2a or IgG2b (all Southern Biotech) were used to generate a standard curve. Goat-anti-mouse IgE-AP, IgG1-AP, IgG2a-AP or IgG2b-AP (all Southern Biotech) were used for detection. For detection of parasite-specific antibodies, plates were coated with 20 μg/mL of HES suspension overnight at 4°C. After blocking for 2 hrs with 3% BSA, samples were incubated at room temperature for 2 hrs and the AP-coupled antibodies named above were used for detection. Absorption was measured on a Multiscan FC photometer (Thermo Fisher) at 405 nm and blank wells were used to subtract background.
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9

SARS-CoV-2 S Protein ELISA for Antibodies

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Ninety-six-well microtiter plates (Nunc MaxiSorp; ThermoFisher Scientific) were coated with 100 μL of recombinant SARS-CoV-2 S protein (Wuhan strain) at a concentration of 1 μg/mL in PBS (Gibco) at 4 °C overnight; negative control wells were coated with 1 μg/mL of BSA (Sigma). Plates were blocked for 1.5 h at room temperature with 280 μL of blocking solution (PBS supplemented with 0.05% Tween-20 (Sigma) and 10% FBS (Corning)). Serum from mice and hamsters were diluted serially in blocking solution, starting at 1:100 dilution and incubated for 1.5 h at room temperature. The plates were washed three times with T-PBS (1X PBS supplemented with 0.05% Tween-20), and 100 μL of goat anti-mouse IgG (Southern Biotech Cat #1030–05) diluted 1:2,000 in blocking solution or 100 μL of HRP-conjugated anti-hamster IgG(H+L) antibody (Southern Biotech Cat. #6061–05) diluted 1:500 in blocking solution, was added to all wells and incubated for 1 h at room temperature. Plates were washed 3 times with T-PBS and 3 times with 1X PBS, and 100 μL of 1-step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to all wells. The reaction was stopped after 5 min using 100 μL of 1M HCl, and the plates were analyzed at a wavelength of 490 nm using a microtiter plate reader (BioTek).
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10

Western Blot Analysis of AKT1

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Cells were seeded in 6-well-plates (2.5 x 105/well) and allowed to adhere for 24h. Whole-cell lysates were prepared using RIPA lysis-buffer containing protease inhibitor (1x, Sigma-Aldrich, St. Louis, Missouri, USA) and phosphatase inhibitor (1x, Merck Millipore, Darmstadt, Germany) and analyzed by Western blot as previously described (22 (link)). The primary antibody rabbit anti-AKT1 (#2938, Cell Signaling Technology) was used for specific AKT1-detection and the mouse anti-β-Actin (sc-47778, Santa Cruz Biotechnology) was used as loading control. Immunocomplexes were detected using peroxidase-conjugated mouse anti-rabbit IgG (sc-2357, 1:8000, Santa Cruz Biotechnology) and goat anti-mouse IgG (1030-05, 1:8000, Southern Biotech) followed by chemiluminescence detection (Westar Nova 2.0, Cyanagen Srl, Bologna, Italy) and Fuji Medical X-Ray Film (Fuji-film Corporation).
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