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Alkaline phosphatase conjugated goat anti mouse igg

Manufactured by Jackson ImmunoResearch
Sourced in France, Cameroon, United States

Alkaline phosphatase-conjugated goat anti-mouse IgG is a secondary antibody used in immunoassays and other immunochemical techniques. It consists of goat-derived antibodies that specifically recognize and bind to mouse immunoglobulin G (IgG), with an alkaline phosphatase enzyme conjugated to it. The function of this product is to detect and amplify the signal of mouse IgG in various applications.

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9 protocols using alkaline phosphatase conjugated goat anti mouse igg

1

Histone-Specific Antibody Secretion Assay

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Multiscreen cellulose filter plates (Millipore) were coated overnight with 10 μg of histone from calf thymus (Sigma-Aldrich). Cells from the mLN were collected and plated starting at 3 × 106/well and in 2-fold serial dilutions in complete RPMI 1640 containing 10% FBS. After 5 h, the wells were washed with PBS containing 0.5% BSA and 0.05% Tween 20, and IgG was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson immunoresearch). Spots were counted using a dissecting microscope. Images were taken using a CTL-immunoSpot S5 analyzer.
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2

Dengue Virus Neutralization Assay

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Concentrations of dengue nAb in Vero cells were measured by PRNT as published [34 (link)]. The CV-1-Fc cell PRNT assays were performed similarly. In brief, identical concentrations of virus for each serotype were used in parallel CV-1-Fc and Vero PRNTs and mixed 1:2 with 2-fold serial dilutions of sera for a final concentration range of 1:10 to 1:20480. Duplicate wells of each sample were evaluated when sufficient sera were available. After 6 days, cell monolayers were fixed and incubated with DENV serotype-specific mouse antienvelope mAbs from Biotem (France): anti-DENV1 (D2-1F1-3), anti-DENV2 (3H5-1-12), anti-DENV3 (8A1-2F12), and anti-DENV4 (1H10-6-7), followed by incubation with alkaline phosphatase-conjugated goat antimouse IgG (Jackson ImmunoResearch). Plaques were visualized with SIGMAFAST BCIP/NBT alkaline phosphatase substrate in levamisole solution. Dengue virus plaques in CV-1-Fc cells for each serotype were typically smaller than plaques from parallel Vero assays but had the shape and density consistent with Vero DENV plaques (data not shown). The number of plaques was counted in high-resolution images of each well.
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3

Measuring Anti-Gag Antibody Response

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Anti-Gag antibody production was measured by ELISA assay. HIV-1 p55 Gag protein (10 µg/ml) was coated onto 96-well ELISA plates overnight at 4°C. Mice serum at different dilutions (1∶30, 1∶120, 1∶480 and 1∶1920) was added to the plates and incubated at room temperature for 2 h with shaking. Gag specific IgG antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson Immunoresearch Inc.). Signal was developed using BluePhos substrate (KPL, Inc.). Plates were analyzed using a 96-well plate absorbance reader at 650 nm. Endpoint titers were calculated as the highest dilution that remained twice the baseline value.
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4

Quantifying Mouse Serum Immunoglobulins

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Total serum IgG levels were measured by ELISA as previously described [19 ]. In brief, 96-well PVDF plates were coated with anti-mouse IgG at 10μg/ml in 1x PBS. Serum samples were added to plates blocked with 1% BSA in 1x PBS. Plates were then washed and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The plates were read with an automated ELISA reader (Molecular Devices, Sunnyvale, CA). Total IgE levels were determined with the BD OptEIA mouse IgE ELISA Set (BD Biosciences, San Jose, CA). For isotype-specific anti-goat Ab levels, plates were coated with goat IgG (10μg/ml) and binding detected with AP-conjugated anti-mouse immunoglobulin isotype Abs.
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5

Viremia and Leukocyte Monitoring in BVDV Infection

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Post-challenge, blood samples were collected in vacutainer tubes containing EDTA on day 0, 2, 3, 6, 9, 13, and 15 for evaluation of viremia and white blood cell (WBC) counts. Blood samples were lysed by freeze-thawing, centrifuged, and the lysate was used for BVDV isolation to determine viral titer by alkaline phosphatase monolayer immunostaining as previously described (6 (link), 44 (link), 45 (link)). Briefly, serial 10-fold dilutions of sample lysate were prepared in Dulbecco’s modified Eagle medium (DMEM) and 50 μl were added to 96-well plate containing fresh MDBK cells. Following incubation at 37°C for 72 h, the cells were fixed for staining with anti-BVDV E2 mAb (Cat. # 348, VMRD) and alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Cat #115-055-146). BVDV titers in blood samples were reported as the lowest dilution at which the lysate-exposed MDBK cells stained positive for BVDV E2. The profile of CBC in each blood sample was evaluated by HESKA Veterinary Hematology System (RTI LLC, Brookings, SD 57006, USA) with counting parameters set for bovine WBC, HGB, RBC, and PLT. The counts for platelets, WBC, and RBC and WBC morphology were verified microscopically.
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6

Quantifying Collagen Types in OA Cartilage

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Five full thickness OA and five control cartilage samples were powdered under liquid N2, then extracted in 4 M guanidine HCl, 0.05MTris/HCl, pH 7.5, at 4°C for 24 h to remove proteoglycans and other matrix proteins. The residues were stored at −20°C for pepsin digestion. Collagen was solubilized by pepsin in 3% (v/v) acetic acid for 24 h at 4°C and then run on SDS-6%PAGE to resolve collagen types II and III for quantitation by densitometry.
SDS-polyacrylamide electrophoresis was run according to Laemmli17 (link) with 6% and 12.5% gels. Proteins bands were stained with Coomassie Brilliant Blue or transblotted to PVDF membrane for Western blot analysis, using the anti-type II and anti-type III collagen-specific mouse monoclonal antibodies. mAb 1C10 binds to a sequence-specific epitope in the denatured α1(II) triple-helical domain (res 934–945)18 (link) and mAb 4G9 binds to a conformational epitope in the collagen type III N-propeptide globular domain8 (link). Westerns were developed after primary antibody incubation using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Avondale, PA) and 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium as substrate for alkaline phosphatase.
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7

Quantification of Serum IgG and IgE Antibodies

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Blood samples were taken at days -21, 20 and 44. To assay serum IgG specific for IL-33, plates were coated with mouse recombinant IL-33 (Fig 1A) and incubated with serum diluted 2500-fold and thereafter with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., PA, USA). For the measurement of HDM-specific IgG and IgE antibodies, plates were coated with the HDM preparation (50 μg protein/ml) [20 (link)] and subsequently incubated with serum (diluted 1:5), followed by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG and IgE.
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8

Whole-Cell ELISA for Bacterial Surface Antigens

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A whole-cell enzyme-linked immunosorbent assay was performed using monoclonal antibodies 6E4 and 3F11 by the method of Abdillahi and Poolman (37 (link)). Bacteria were grown on sRPMI plates, which were supplemented with Neu5Ac where indicated. Cells were harvested and resuspended in phosphate-buffered saline (PBS) to an optical density at 600 nm of 0.10. Microtiter plate wells were filled with 100 µl of the bacterial suspension and dried at 40°C overnight. The wells were washed, and ELISA was performed with MAb 6E4 at a dilution of 1:100. Secondary antibody (alkaline phosphatase-conjugated goat anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA) was used at a dilution of 1:10,000. The substrate p-nitrophenylphosphate was applied at a concentration of 1.0 mg/ml. Absorbance was read in a Tecan Model Infinite 200 Pro plate reader at 405 nm.
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9

Histone-Specific Antibody Secretion Assay

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Multiscreen cellulose filter plates (Millipore) were coated overnight with 10 μg of histone from calf thymus (Sigma-Aldrich). Cells from the mLN were collected and plated starting at 3 × 106/well and in 2-fold serial dilutions in complete RPMI 1640 containing 10% FBS. After 5 h, the wells were washed with PBS containing 0.5% BSA and 0.05% Tween 20, and IgG was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson immunoresearch). Spots were counted using a dissecting microscope. Images were taken using a CTL-immunoSpot S5 analyzer.
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