The IgG anti-β2GPI antibodies were determined by ELISA as previously described [[7] (link), [8] (link), [9] (link)]. In brief, micro-titer plates (Maxisorp 269787; Thermo Scientific Nunc) were coated overnight to 4 °C with a solution containing 25 μg/mL of β2GPI purified in the laboratory as was previously described or by β2GPI reference (Donate by S.S. Pierangeli PhD, Division of Rheumatology University of Texas Medical Branch) and plates were blocked with 3% BSA (sufficient concentration to obtain a good blocking of the plate). Samples and controls (diluted 1:50 in 3% BSA solution) were added to the plates in triplicate and incubated by 1 h, pooled normal mouse plasma was used as negative controls. Plates were washed three times with PBS-Tween 20 0.5% pH 7.2 and incubated with alkaline-phosphatase anti-mouse IgG (Sigma-Aldrich, MO, USA). The color was developed by the addition of 1 mg/ml of p-nitrophenolphosphate (Sigma-Aldrich, MO, USA), and the reaction was stopped by the addition of 3 N NaOH. Then the plate was read in a Biotek ELx800 (Biotek, VT, USA) ELISA reader at 405 nm. A sample was considered positive when its OD was greater than 3 standard deviations (SD) from the average of normal controls (cut-off).
Anti mouse igg alkaline phosphatase
Manufactured by Merck Group
Sourced in United States
Anti-mouse IgG-alkaline phosphatase is a laboratory reagent used to detect and quantify the presence of mouse immunoglobulin G (IgG) in samples. It consists of alkaline phosphatase, an enzyme, conjugated to antibodies that specifically bind to mouse IgG. This conjugate can be used in various immunoassay techniques to measure mouse IgG levels in biological samples.
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Lab products found in correlation
Anti rat igg alkaline phosphatase, by Merck Group (2 mentions)
P nitrophenyl phosphate, by Merck Group (2 mentions)
Fast track 2.0 kit, by Thermo Fisher Scientific (2 mentions)
Incomplete freund s adjuvant, by Merck Group (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Elx800, by Agilent Technologies (1 mentions)
P nitrophenol phosphate, by Merck Group (1 mentions)
Mil 2, by Thermo Fisher Scientific (1 mentions)
Protein free blocking buffer, by Thermo Fisher Scientific (1 mentions)
Bcip nbt phosphatase substrate, by Merck Group (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
Alkaline phosphatase conjugate substrate kit, by Bio-Rad (1 mentions)
Mouse anti ha antibody, by BioLegend (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Anti dcp1a, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg peroxidase, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Storm 860, by GE Healthcare (1 mentions)
Anti flag, by Merck Group (1 mentions)
25 protocols using anti mouse igg alkaline phosphatase
1
ELISA Assay for Anti-β2GPI Antibodies
2
Quantifying IgG-secreting B Cells in Mice
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After activation with 35% ethanol, 96-well polyvinylidene difluoride plates (MAIP4510, Millipore) were coated with 70 μl per well of 5 μg ml−1 mIL-2 (Peprotech) overnight at 4 °C. After washing with PBS, plates were blocked with Protein-Free Blocking Buffer (Thermo) for 1 h at room temperature and then with complete RPMI medium for 30 min at room temperature. Serially diluted spleen or bone marrow cells (5 × 104 to 4 × 105 cells per well in complete RPMI medium) from 10- to 18-week-old female B6 or NOD mice were added in the ELISPOT plate. In a set of experiments, splenocytes from 10- to 18-week-old female B6 or NOD mice were cultured for 6 days at 1 × 106 cells ml−1 in complete RPMI medium with 10 μg ml−1 CpG-ODN 1018 to allow expansion of memory B cells. Serially diluted CpG pre-activated splenocytes (5 × 104 to 4 × 105 cells per well) were then added in the ELISPOT plate. After a 18 h culture, plates were washed three times with PBS/0.25% Tween-20, three times with PBS and then incubated with alkaline phosphatase-anti-mouse-IgG (1:1000; Sigma-Aldrich) diluted in PBS/2% BSA for 2 h at room temperature. Plates were then washed and phosphatase activity measured adding 100 μl per well substrate (Bio-Rad). Reaction was blocked by extensive washing with tap water after 15 min incubation. Spots were counted with an AID camera.
3
Quantifying Antigen-specific Antibody-secreting B Cells
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The Enzyme-Linked ImmunoSpot assay (ELISpot) was conducted to quantify antigen-specific antibody-secreting B cells (ASC). The PVDF membranes of ELISpot plate were activated by incubating in 35% ethanol for 10 min at room temperature. The plate was washed thrice with PBS buffer to remove residue ethanol, and 500 ng/well of bacterial antigen (bCOE) added, in PBS buffer at 4°C overnight. Following washing, 200 µl of complete RPMI containing 10% FBS was added and plates incubated at 37°C, 5% CO2, for 1 h, to prevent non-specific binding. Splenocytes were isolated from mice 10 days after final immunization using Percoll density gradients. Viable splenocytes were counted by trypan blue method (Thermo Fisher Scientific). Cells were diluted in complete RPMI medium to 1 × 106, 5 × 105, and 1 × 105 cells/well and then cultured at 37 °C, 5% CO2 for 2 days. After removal of unbound cells by washing, anti-mouse IgG alkaline phosphatase (Sigma) was added and following 2 h incubation at 37°C, the cells were washed three times as before. ASC were visualized by addition of BCIP/NBT phosphatase substrate (Sigma) and colour spots counted under microscope and computer.
4
Protein Isolation and Western Blotting
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Protein samples (~1 mg) were isolated from the dorsal part of the thorax. Western blotting was carried out by using an SDS-containing gradient acrylamide gel, nitrocellulose blotting membrane, and TBST washing solution. In addition, 3% milk powder solved in TBST was used. Proteins were labeled at 4 °C overnight by the following primary antibodies: anti-ubiquitin (mouse, 1:500, Merck, Rahway, NJ, USA, ST1200), anti-Atg8a (rabbit, 1:2000 [64 (link),65 (link)]), anti-p62-vel (rabbit, 1:2000 [56 (link)]), anti-α-Tub84B (mouse, 1:2500, Sigma, St. Louis, MO, USA, T6199) and anti-EDTP (rat, 1:500 [19 (link)]). The following secondary antibodies were used (at room temperature for an hour): anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rat IgG alkaline phosphatase (1:1000, Sigma, A8438). Primary and secondary antibodies were washed out 3 times for 10 min in TBST, and finally, membranes were incubated in an AP buffer. NBT/Bcip (Sigma, 72091) was used for recording the antibody staining, and NBT/Bcip was dissolved in an AP buffer (1:50).
5
Recombinant ALDH10s Expression in E. coli
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The recombinant Arabidopsis thaliana [L.] Heynh. ALDH10s were induced in transformed Escherichia coli strain BL21 cells (EMD Millipore) by the addition of isopropyl β-D-1-thiogalactopyranoside (final concentration of 0.4 mM) to the Lysogeny broth medium containing 50 μg mL−1 ampicillin when the cell culture had an OD600 of 0.5. The cells were collected 4 h after induction by centrifugation at 5000× g for 5 min and then stored at −80 °C. Bacterial lysis and purification of the recombinant protein by affinity chromatography were conducted as described previously7 (link). High-protein fractions were combined and precipitated on ice by slowly adding solid ammonium sulfate, with gentle stirring, to 80% saturation. Aliquots of precipitated protein were stored at −80 °C. Total proteins were separated by SDS-PAGE gel electrophoresis and visiualized by staining with Coomassie Blue R-250 using standard protocols33 (link). Immunoblot analysis was based on a semi-dry method using a mouse monoclonal IgG against the His tag (Santa Cruz Biotechnology, 1:1000) and an anti-mouse IgG–Alkaline Phosphatase (Sigma 1:10000) as primary and secondary antibodies, respectively. Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit was used to detect fusion proteins.
6
Quantifying IL-5 Nanobody Binding
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Human IL-5-Fc, cynomolgus IL-5-Fc and mouse IL-5-Fc were expressed and purified in HEK 293F cells. 5 μg/mL proteins (human IL-5-Fc, cynomolgus IL-5-Fc, mouse IL-5-Fc and IgG1) were coated onto microtiter plates at 4 °C overnight. After blocking with 1% bovine serum albumin (BSA) for 2 h, 10 μg/mL IL-5 Nb was added and incubated at room temperature for 1 h. Next, the plates were incubated with mouse anti-HA antibody (Biolegend, San Diego, CA, USA), followed by anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich). The absorbance at 405 nm was read on a microplate reader (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of Protein Markers
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The cells were washed with PBS and scraped after the addition of a buffer solution (160 mM Tris-HCl; pH 6.8; 4% SDS; 10% b-mercaptoethanol; 24% glycerol and 0.02% bromophenol blue) that promotes cell lysis and denatures proteins. The cell extract was collected, incubated at 95 °C for 10 min and subjected to SDS-PAGE. Then, the proteins were transferred to nitrocellulose membranes. The nitrocellulose membrane was blocked (1-h incubation with 5% milk in Tris-buffered saline—TBS), washed and incubated with the primary antibodies (anti-DDX6, MBL, Japan, PD009, 1:2500; anti-DCP1A, Santa Cruz, Dallas, TX, USA, sc-100706, 1:200 and anti-RPL30, Abcam, Cambridge, UK, ab170930). After incubation with the suitable secondary antibodies (anti-mouse IgG-alkaline phosphatase, Sigma, St. Louis, MO, USA, 1:1000 and anti-rabbit IgG-peroxidase, produced in goat, Sigma, St. Louis, MO, USA, 1:2500), the membranes were analyzed with AP buffer, BCIP e NBT (Promega, Madison, WI, USA) or with a Novex® ECL HRP chemiluminescent kit (Invitrogen, Carlsbad, CA, USA). The signal intensity was quantified with ImageJ software [56 (link)].
8
Quantifying Protein Expression in Cell Lysates
9
Protein Expression Analysis in Drosophila Heads
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Western blot samples were prepared from 10 female heads, which were treated in 32 μl of Fly Lysis buffer + 32 μl 2 × Laemmli buffer. 15 µl samples were run on 4–20% Mini-PROTEAN® TGX™ Gel and blotted onto Nitrocellulose Membrane (Kisker Biotech, 40520100). After blocking with 3% Milk Powder (BioRad 170-6404 /Blotting-Grade Blocker/) dissolved in TBST, membranes were probed with specific antibodies [anti-Tubulin (1:1000, mouse, Sigma T6199), anti-Ref(2)P (1:2000, rabbit48 (link)), anti-Atg8a (1:2500, rabbit50 (link)), anti-EDTP, 1:1000, rat22 (link), anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-rat IgG alkaline phosphatase (1:1000, Sigma, A5153), and developed by NBT-BCIP solution (Sigma, 72091). Each western blot analysis was repeated at least three times with independent biological samples.
10
Serum PE-specific IgG Quantification by ELISA
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Serum PE-specific total IgG titers were measured by ELISA. Briefly, maxisorp 96-well plates (Nunc) were coated with a PE solution (2.5 μg/ml) in carbonate buffer (100 μl/well) overnight at 4°C. Plates were then blocked by addition of PBS, 3% polyvinylpyrrolidone (PVP) (SERVA) (200 μl/well), incubated for 2 h at 37°C and then washed once with PBS, 0.05% Tween20 (washing buffer). Serial dilutions (3-fold step) of standard and serum samples in PBS, 0.05% Tween20, 1% BSA were added to the wells and incubated for 2 h at 37°C. Plates were then washed 3 times with washing buffer and incubated for 1 h at 37°C with anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich) solution (100 μl/well). After 3 washes the substrate p-nitrophenylphosphate (Sigma-Aldrich) (100 μl/well) was added for 30 min at room temperature. Absorbance at 405 nm was then measured by a plate spectrophotometer (BioTek–ASHI). Antibody titers were expressed as the reciprocal dilution corresponding to a cut-off at OD405 = 0.5.
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