Cell lysates were collected using sample buffer solution with reducing reagent (6×) for SDS/PAGE (Cat. No. 09499‐14; Nacalai Tesque, Kyoto, Japan), then electrophoresed onto a 10% SDS/PAGE, and blotted onto a poly(vinylidene difluoride) membrane. The membranes were incubated with anti‐nephronectin (Cat. No. AF4298; R&D Systems, Minneapolis, MN, USA) and anti‐actin (Cat. No. A5060; MERCK, Darmstadt, Deutchland) as the first antibodies and then further probed with anti‐mouse IgG horseradish peroxidase‐linked (Cat. No. NA931V; GE Healthcare, Little Chalfont, UK) and anti‐goat IgG horseradish peroxidase‐linked (Cat. No. NB7352; NOVUS, Littleton, CO, USA) secondary antibodies. Proteins were visualized using ECL Prime Western Blotting Detection reagent (Cat. No. #RPN2232; GE Healthcare).
Anti mouse igg horseradish peroxidase linked
Manufactured by Merck Group
Sourced in United States
Anti‐mouse IgG horseradish peroxidase‐linked is a laboratory reagent used as a detection system in various immunoassays. It consists of anti-mouse immunoglobulin G (IgG) antibodies conjugated with the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Bio-Rad (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (2 mentions)
A1978 200ul, by Merck Group (2 mentions)
Sc 393232, by Santa Cruz Biotechnology (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Anti actin, by R&D Systems (1 mentions)
3 protocols using anti mouse igg horseradish peroxidase linked
1
Western Blot Analysis of Nephronectin
2
Quantitative Western Blotting for GR
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Protein was extracted and quantified using Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). 20 μg total protein was separated using SDS-PAGE and transferred to PVDF membranes. Blots were blocked in a Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% skim milk for 1 h at RT, then incubated overnight in primary antibodies for GR (1:1000; sc-393,232, SantaCruz Biotechnology, Santa Cruz, CA, USA) and β-actin (1:10,000; a1978-200UL, Sigma-Aldrich, St. Louis, MO, USA), diluted in TBST with 1% skim milk. Blots were washed and probed with secondary antibody for 1 h at RT (anti-mouse IgG, horseradish peroxidase linked; 1:5000; Merck, Burlington, MA, USA). Bands were visualized using high sensitivity Pierce ECL Plus (Thermo Scientific) and quantified using the Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The optical density of GR was normalised to β-actin and a cross-membrane control.
3
Western Blot Analysis of Glucocorticoid Receptor
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Protein was extracted and quantified using Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). 20µg total protein was separated using SDS-PAGE and transferred to PVDF membranes. Blots were blocked in a Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% skim milk for one hour at RT, then incubated overnight in primary antibodies for GR (1:1,000; sc-393232, SantaCruz Biotechnology, Santa Cruz, CA, USA) and β-actin (1:10,000; a1978-200UL, Sigma-Aldrich, St. Louis, MO, USA), diluted in TBST with 1% skim milk. Blots were washed and probed with secondary antibody for one hour at RT (antimouse IgG, horseradish peroxidase linked; 1:5,000; Merck, Burlington, MA, USA). Bands were visualized using high sensitivity Pierce ECL Plus (Thermo Scientific) and quantified using the Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The optical density of GR was normalised to β-actin and a cross-membrane control.
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