DNA methylation levels in BMDMs were detected by dot blot as described before [31 (link)]. DNA was isolated from naive BMDMs using DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) and quantified with Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). DNA was then denatured, neutralized, serial 2-fold diluted and spotted onto an Hybond-N+ nylon membrane (GE Healthcare, Eindhoven, The Netherlands). After drying, the membrane was exposed to UV light for 30 s at 120,000 μJ/cm2 for crosslinking and then blocked in 5% milk TBST for 1 h at room temperature. Thereafter, the membrane was incubated for 1 h at room temperature with monoclonal primary antibodies against m5C (1:1000 dilution, 28692S; Cell Signaling Technology) in 5% milk TBST. After probing with HRP-conjugated secondary antibodies against rabbit IgG (#7074, Cell Signaling Technology), blots were imaged using Lumilight plus ECL substrate (Roche) on an ImageQuant LAS 4000 biomolecular imager (GE Healthcare). The same amount of total DNA (200, 100 and 50 ng) was spotted on another membrane and then was stained with 0.02% methylene blue (Sigma-Aldrich, Amsterdam, The Netherlands) in 0.3 M sodium acetate (pH 5.2).
Hrp conjugated secondary antibodies against rabbit igg
Manufactured by Cell Signaling Technology
HRP-conjugated secondary antibodies against rabbit IgG are laboratory reagents used to detect and quantify rabbit immunoglobulin G (IgG) in various immunoassays. The antibodies are conjugated with horseradish peroxidase (HRP), an enzyme that generates a colorimetric or chemiluminescent signal upon reaction with a suitable substrate, allowing for the visualization and quantification of target rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Hybond n nylon membrane, by GE Healthcare (1 mentions)
Lumilight plus ecl substrate, by Roche (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hek 293a, by Merck Group (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Hek293a cells, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
2 protocols using hrp conjugated secondary antibodies against rabbit igg
1
DNA Methylation Analysis in Macrophages
2
CRISPR-Cas9 Mediated LRRC8A Knockout in HEK293A Cells
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HEK293A cells (Invitrogen) and LRRC8A-knockout HEK293A cells were cultured in DMEM-high glucose (Sigma-Aldrich, Cat. #D5671), supplemented with 10% fetal bovine serum (FBS; BioWest, Cat. #S1560-500) and 100 units/ml penicillin G (Meiji Seika, Cat. #6111400D2039). All cells were cultured in a 5% CO 2 atmosphere at 37ºC and verified to be negative for mycoplasma. LRRC8A-knockout HEK293A cells were established using the CRISPR-Cas9 system 39 . Briefly, the targeting sequence for human LRRC8A, 5′-GCACAACATCAAGTTCGACGTGG-3′, was designed using the CRISPR design tool (http://crispr.mit.edu/), and oligo DNAs for its sense and anti-sense were purchased from Bethyl Laboratories, MBL, and Sigma-Aldrich, respectively.
HRP-conjugated secondary antibodies against rabbit IgG (Cat. #7074, 1:2,000) and mouse IgG (Cat. #7076, 1:10,000-1:20,000) were purchased from Cell Signaling Technology.
HRP-conjugated secondary antibodies against rabbit IgG (Cat. #7074, 1:2,000) and mouse IgG (Cat. #7076, 1:10,000-1:20,000) were purchased from Cell Signaling Technology.
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