Media supernatants from the in vitro plasma cell differentiation assay were measured for secreted IgM by ELISA. 96-well plates (Costar, #3690) were coated with 1 μg/ml goat anti-IgM (Southern Biotech, #1020–01) in PBS. Wash buffer (PBS with 0.05% Tween-20 (v/v)) was used for all washing steps and blocked with PBS-BB (PBS without Ca2+/Mg2+, 1% BSA (w/v), 0.05% Tween-20(v/v)). Supernatants from LPS stimulated samples were diluted (1:100) and media only samples were diluted (1:10) in PBS-BB. IgM standard (Southern Biotech, #0101–01) was prepared in a 2-fold serial dilution in PBS-BB. IgM was detected with Goat anti-IgM-HRP (Southern Biotech, #1020–05) diluted (1:3000) in PBS-BB. ELISA plates were developed with TMB (Sigma) and stopped with 1 N sulfuric acid. Absorbance was measured at 450 nm using spectrophotometer (Spectramax M5, Molecular Devices). Sample IgM concentration values were calculated with SoftMaxPro v7 software.
Goat anti igm hrp
Manufactured by Southern Biotech
Sourced in United States
Goat anti-IgM-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP) that binds to the Fc region of human IgM antibodies. It is used in various immunoassay techniques to detect and quantify the presence of IgM in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Igm standard, by Southern Biotech (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igm, by Southern Biotech (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Tris glycine gel, by Bio-Rad (1 mentions)
Goat anti mouse hrp, by Bio-Rad (1 mentions)
Goat anti rabbit hrp, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mini protean electrophoresis system, by Bio-Rad (1 mentions)
5 protocols using goat anti igm hrp
1
Measuring IgM Secretion in Plasma Cell Differentiation
2
Measurement of Anti-PC and Anti-MDA Antibodies
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Levels of anti-phosphorylcholine (PC) and anti-malondialdeyhyde (MDA) antibodies were measured in duplicate from frozen serum samples by in-house assays, as previously described (Gronwall et al., 2012 (link)). Herein, ELISA wells were coated with PC6-BSA (Biosearch Technologies Inc., Novato, CA, USA) or MDA-BSA (Academic Bio-medical Co., Houston, TX, USA). Values were reported from 1:1000 dilutions in 1% BSA-PBS after detection with goat anti-IgM-HRP (Southern Biotech, Birmingham, AL, USA) or goat anti-IgG-HRP (Jackson Immunosearch, West Grove, PA, USA). An established standard curve from a SLE pool was used for calibration. Assays for IgG anti-PC or IgM anti-MDA were not included as pilot studies have shown these levels to be non-informative [data not shown] (Gronwall et al., 2012 (link)).
3
Measuring IgM Secretion in Plasma Cell Differentiation
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Media supernatants from the in vitro plasma cell differentiation assay were measured for secreted IgM by ELISA. 96-well plates (Costar, #3690) were coated with 1 μg/ml goat anti-IgM (Southern Biotech, #1020–01) in PBS. Wash buffer (PBS with 0.05% Tween-20 (v/v)) was used for all washing steps and blocked with PBS-BB (PBS without Ca2+/Mg2+, 1% BSA (w/v), 0.05% Tween-20(v/v)). Supernatants from LPS stimulated samples were diluted (1:100) and media only samples were diluted (1:10) in PBS-BB. IgM standard (Southern Biotech, #0101–01) was prepared in a 2-fold serial dilution in PBS-BB. IgM was detected with Goat anti-IgM-HRP (Southern Biotech, #1020–05) diluted (1:3000) in PBS-BB. ELISA plates were developed with TMB (Sigma) and stopped with 1 N sulfuric acid. Absorbance was measured at 450 nm using spectrophotometer (Spectramax M5, Molecular Devices). Sample IgM concentration values were calculated with SoftMaxPro v7 software.
4
Mouse IgM Binding ELISA Protocol
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High-affinity protein binding microplates (Corning) were coated overnight goat anti-mouse IgM (1mg/ml, Southern Biotech, cat# 1021–01) and blocked with coating buffer containing with 1% BSA (w/v) and 2% goat serum (Omega Scientific) in PBS. Wells were washed with ELISA wash buffer (1X PBS, 0.05% Tween-20). Mouse serum samples were diluted 1:400 in coating buffer and incubated in the wells for 1 hr. Wells were washed 5 times and secondary goat anti-IgM-HRP (Southern Biotech, cat# 1021–08) at 1:5000 dilution was incubated for 1 hr in the wells. Wells were washed several times and developed with TMB substrate (Invitrogen). Development was stopped after 20 minutes with 1M H3PO4 stop solution. Absorbance was measured at 450nm on a BioTek Epoch microplate spectrophotometer.
5
SDS-PAGE and Western Blot Analysis
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