The mAbs used for flow cytometric analysis and other experiments were obtained from eBioscience (San Diego, CA, USA) or BD Biosciences (San Diego, CA, USA): FITC-labeled anti-mouse CD4 (RM4-5), CD45 (30-F11); PE-labeled anti-mouse IFN-γ (XMG1.2), FoxP3 (FJK-16s); PerCP-labeled anti-mouse CD4 (L3T4, RM4-5); allophycocyanin (APC)-conjugated anti-mouse IL-17A (eBio17B7); biotin-conjugated anti-mouse IL-4 (BVD6-24G2), and streptavidin-conjugated PerCP. PMA and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Abs used to detect OVA-specific-IgE, IgG, IgG1, and IgG2a levels in sera were purchased from Southern Biotech (Birmingham, AL, USA): unconjugated (human absorbed) anti-mouse IgG, goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgE, HRP-conjugated goat anti-mouse IgG, goat anti-mouse IgG1, goat anti-mouse IgG2a, and rat anti-mouse IgE-HRP. The primers specific for cytokines, mucins, and tight junctions (TJs) were synthesized by Bioneer Corp. (Daejeon, Korea) and used for PCR amplification of target genes.
Goat anti mouse igg2a
Manufactured by Southern Biotech
Sourced in United States
Goat anti-mouse IgG2a is a secondary antibody that specifically recognizes the IgG2a subclass of mouse immunoglobulins. It is commonly used in immunological techniques such as ELISA, Western blotting, and immunohistochemistry to detect and quantify the presence of mouse IgG2a in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg1, by Southern Biotech (8 mentions)
Goat anti mouse igg2b, by Southern Biotech (3 mentions)
Goat anti mouse iga, by Southern Biotech (3 mentions)
Goat anti mouse igm, by Southern Biotech (2 mentions)
Rabbit anti goat igg conjugated with horseradish peroxidase, by Southern Biotech (2 mentions)
Goat anti mouse igg conjugated with horseradish peroxidase, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (1 mentions)
Goat anti mouse ige, by Southern Biotech (1 mentions)
Rat anti mouse ige hrp, by Southern Biotech (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Diethanolamine, by Merck Group (1 mentions)
Ap conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by Southern Biotech (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Goat anti mouse ig, by Southern Biotech (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Necrostatin 1, by Enzo Life Sciences (1 mentions)
15 protocols using goat anti mouse igg2a
1
Antibody-based Immunological Analyses
2
ELISA for Virus-Specific Antibody Titers
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Purified β-propiolactone-inactivated rWT virus (2.5 μg/mL) was coated onto 96-well Immulon-2 plates (Dynex Technology Inc., Chantilly, VA, USA) and incubated overnight at 4°C. Serially diluted mouse sera was added and left to incubate at room temperature for 1.5 h. Goat anti-mouse IgG (H+L) (Invitrogen, B2763, Carlsbad, CA, USA), goat anti-mouse IgG1 (Southern Biotech, 1070-08, Birmingham, AL, USA), or goat anti-mouse IgG2a (Southern Biotech, 1080-08, Birmingham, AL, USA) was added for 1 h at room temperature to detect IgG, IgG1, or IgG2a, respectively. Color development was initiated by the addition of alkaline phosphatase (AP)-conjugated streptavidin (Jackson ImmunoResearch) and p-nitrophenyl phosphate (PNPP) substrate [10 mg/mL p-nitrophenyl phosphate di (tris) salt crystalline (Sigma-Aldrich, St. Louis, MO, USA), 1% diethanolamine (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL MgCl2, pH 9.8]. Detection of the optical density (OD) occurred at 405 nm (using a reference filter of 490 nm) on a microplate reader (Molecular Devices SpectraMax Plus 384, San Jose, CA, USA). The titres of each sample were determined as the highest dilution at which the OD of the sample was larger than the defined cut-off. The cut-off was defined as the mean OD of a known negative sample plus twice the standard deviation.
3
Bat Serum IgG Assay Development
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Sera from several distinct Megachiropteran bat species were purchased through cooperation with the Lubee Bat Conservancy (Gainesville, FL). Purified IgG derived from dog, cat, guinea pig, swine, hamster, human were purchased (Rockland Immunochemicals). Laboratory stocks of mouse and rat mAbs were used as unlabeled IgG or conjugated with EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific); horse serum was obtained from the Wadsworth Center Veterinary Sciences facility. For ELISAs, unlabeled and horseradish peroxidase (HRP)- conjugated goat anti-mouse Ig, goat anti-mouse IgG1, goat anti-mouse IgG2a, and goat anti-mouse IgM, as well as HRP-streptavidin were used (Southern Biotech). In addition, polyclonal HRP-conjugated goat anti-bat Ig was purchased (Bethyl Laboratories). For flow cytometry, rabbit anti-mouse Ig (RamIg) (Jackson Research, West Grove, PA) was purchased and labeled with FITC (Sigma-Aldrich) or Cy-5 (GE HealthCare).
4
Western Blot Analysis of Cell Death Regulators
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Antibodies with the following specificities were used for Western blot analysis: TRAF2, β-actin (abcam, Cambridge, UK), Caspase-3, cIAP1, cIAP2 (Cell signaling, Danvers, MA, USA), Caspase-8 (Enzo Life Sciences, Lausen, Switzerland), PARP, Cul3 (BD Pharmingen, Heidelberg, Germany), α-Tubulin (Millipore, Billerica, MA, USA) and RIP3 (Imgenex, San Diego, CA, USA). HRP-conjugated goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b and donkey anti-rabbit antibodies were obtained from Southern Biotech (Birmingham, AL, USA) and Dianova (Hamburg, Germany). Killer-TRAIL was purchased from Alexis biosciences (Carlsbad, CA, USA). The pan-caspase-inhibitor zVAD-fmk was obtained from Bachem (Heidelberg, Germany). Necrostatin-1 was purchased from Enzo Life Sciences (Lausen, Switzerland). Fc-CD95L and Flag-TWEAK were produced as previously described.33 (link) TRAF2 siRNA (HS-TRAF2_4, sequence as follows: 5′-GGACCAAGACAAGAUUGAATT-3′), alternate TRAF2 siRNA's (HS-TRAF2_5, sequence: 5′-CGAGGGCAUAUAUGAAGAATT-3′ HS TRAF2_6, sequence: 5′-GUUCGGCCUUCCCAGAUAATT-3′ HS TRAF2_7, sequence: 5′-GCUGCGGAGCAGACGUGAATT-3′) and negative control siRNA were provided by Qiagen (Hilden, Germany). siRNA transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
5
Antibody Response Evaluation by ELISA
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Seven days after last immunisation, the sera of anti-rMntC and anti-immunodominant peptide were collected from the tail vein for analysis of IgG and other antibodies using ELISA. The assay of antibody detection by using ELISA was as described previously[21 (link)]. The corresponding collected serum samples from mice immunised with rMntC and epitope vaccination were employed as the primary antibodies and the appropriate horseradish peroxidase-conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany), or goat anti-mouse IgG1, or goat anti-mouse IgG2a, or goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) were used as the secondary antibody. The highest absorbance was calculated by log test from the reduplicate assay and blank control and the comparison between the two results was undertaken by t-test.
6
ELISA Assay for Anti-Aβ42 Antibody Characterization
7
ELISA Protocol for PspA4Pro Antibody
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ELISA was carried out as described previously [12 (link)] in plates coated with 5 μg/ml PspA4Pro. For the detection of serum antibodies, goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma-Aldrich) was used as secondary antibody. For IgG isotyping, goat anti-mouse IgG1, goat-anti-mouse IgG2a and rabbit anti-goat IgG conjugated with horseradish peroxidase (Southern Biotech) were used. The titer was defined as the reciprocal of the highest dilution with an A492 ≥ 0.1. For the detection of antibodies in BALF, goat anti-mouse IgG conjugated with alkaline phosphatase (AP), goat anti-mouse IgA and rabbit anti-goat IgG conjugated with AP (Southern Biotech) were used and A405 was measured. Background value of each plate (0.040 to 0.045) was subtracted from the actual absorbance obtained for each sample.
8
ELISA Protocol for Antibody Detection
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Immulon 4HBX 96-well plates (Fisher, 14–245-153) were coated with 50 μL protein antigens at 2μg/ml in PBS and incubated overnight at 4°C. Plates were washed three times and blocked for one hour with 125 μL of blocking buffer (PBS with 3% goat serum, 0.5% nonfat milk, 0.1% Tween 20). Two-fold serial dilutions of serum samples were made in blocking buffer starting at a 1:50 dilution. Plates were washed three times, then diluted serum was added to plates and incubated for two hours. Detection antibodies conjugated to HRP were diluted in blocking buffer: rabbit anti-mouse IgM (Jackson Immunoresearch, 315–035-049) diluted 1:5000; goat anti-mouse IgK (Novus, NB7549) diluted 1:5,000; goat anti-mouse IgG1 (Southern Biotech, 1071–05) diluted 1:5,000; goat anti-mouse IgG2a (Southern Biotech, 1081–05) diluted 1:2,500. Plates were washed three times and diluted detection antibodies were added to plates and incubated for one hour. Plates were washed three times and 50μL of room temperature KPL SureBlue TMB substrate was added to wells. Plates incubated for 5 minutes before quenching with 25μL of 250mM HCl. Well absorbance was read at 450 nm. All wash steps were conducted using PBS with 0.1% Tween 20.
9
Serum Antibody Detection by ELISA
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ELISA was performed as described by Moreno et al. [24 (link)] using 96-well microplates coated with 5 μg/mL PspA4Pro. For the detection of serum antibodies, goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma-Aldrich, Gillingham, UK) was used as secondary antibody. For IgG isotyping, goat anti-mouse IgG1, goat-anti-mouse IgG2a, and rabbit anti-goat IgG conjugated with horseradish peroxidase (Southern Biotech) were used. The titer was defined as the reciprocal of the highest dilution with an absorbance at 492 nm ≥ 0.1.
10
Murine Antibody Response to Recombinant E. coli Proteins
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Serum, bronchoalveolar lavage (BAL), stool (GUT, gastrointestinal tract secretions) and vaginal lavage (VAL) from mice orally immunized with E. coli expressing recombinant proteins were tested by indirect ELISA for the presence of IgG and IgA. IgG was further isotyped for IgG1 and IgG2a. Purified recombinant 6xHis-tagged proteins were coated at 2 μg/ml on Nunc MaxiSorp™ flat-bottom ELISA plates (eBioscience, San Diego, CA, USA) and indirect ELISA was performed using serum (1:100), BAL (1:1), VAL (1:1) and GUT (1:1). Goat anti-mouse IgG (1:50,000), Goat anti-mouse IgG1 (1:50,000), Goat anti-mouse IgG2a (1:50,000) and goat anti-mouse IgA (1:50,000) horseradish peroxidase-conjugated antibody (SouthernBiotech, Birmingham, Alabama) was used as secondary antibody.
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