Western blotting was performed as described earlier [22 (link), 23 (link)]. ln brief, 1 × 106 cells each were lysed in pre-cooled RIPA buffer (Pierce, USA) containing phosphatase and proteinase inhibitors and 2.5 mmol/l dithiothreitol (Sigma-Aldrich). Equal amounts of proteins (30 μg) were loaded on a 10 % polyacrylamide gel (SDS-PAGE), electrophoresed, and then blotted by semi-dry transfer onto a nitrocellulose membrane (Schleicher & Schuell, Germany). After a blocking step with 5 % non-fat milk (Merck, Germany), membranes were incubated with either a rabbit anti-DT diaphorase primary antibody (NQ01, N5288, Sigma-Aldrich; diluted 1:2,000) or a rabbit anti-LC3-I/-II primary antibody (AHP2167T; AbD Serotec GmbH, Germany; diluted 1:1,000). After washing with phosphate buffered saline (PBS), membranes were incubated with a horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (KPL, USA; diluted 1:10,000) for 60 min at room temperature. A monoclonal mouse anti-β-actin primary antibody (Abcam, UK; diluted 1:10,000) was used as loading control and visualized with the goat anti-mouse secondary antibody (KPL, diluted 1: 10,000). lmmunoblots were visualized by enhanced chemiluminescence western blotting substrate (Pierce, Thermo Scientific, USA) with subsequent exposure on an X-ray film (Fuji Super RX medical X-ray films; Fuji, Germany) for 30 s.
Fuji super rx medical x ray films
Manufactured by Fujifilm
Sourced in Germany
Fuji Super RX medical X-ray films are designed for use in medical imaging. They are intended for the capture and processing of X-ray images.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (1 mentions)
Goat anti mouse secondary antibody, by Abcam (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Non fat milk, by Merck Group (1 mentions)
Hrp linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
3 protocols using fuji super rx medical x ray films
1
Western Blotting Protein Quantification
2
Western Blotting and Collagen IV Analysis
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Laemmli sample buffer was added to the cell lysate samples and these were run on 8–10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were blocked in phosphate buffered saline containing 0,1% Tween- 20% and 5% dry milk. The first antibodies were diluted in phosphate buffered saline containing 0,1% Tween- 20% and 5% bovine serum albumin and used either for 2 h at room temperature or overnight at 4 °C. After several washing steps in PBS-Tween-20, membranes were incubated with HRP-linked secondary antibodies (Santa Cruz Biotechnology, Inc., USA) diluted in blocking buffer. Antibody binding was detected using enhanced chemiluminescence and Fuji Super RX medical X-ray films.
For the detection of collagen IV NC1 monomers and dimers, cells or small tissue pieces were harvested in hypotonic lysis buffer (10 mM CaCl2, 50 mM Hepes, pH 7.4) completed with 0.1 mM benzamidine hydrochloride, 25 mM 6-aminocaproic acid, 1 mM Phenylmethylsulfonyl fluoride and 0.5 mg/ml type I collagenase at 37 °C for 12–18 h. For the lysis of hypoxia/anoxia treated and control cells, the lysis buffer was supplemented also with 150 µM phloroglucinol to inhibit PXDN activity during the lysis process.
For the detection of collagen IV NC1 monomers and dimers, cells or small tissue pieces were harvested in hypotonic lysis buffer (10 mM CaCl2, 50 mM Hepes, pH 7.4) completed with 0.1 mM benzamidine hydrochloride, 25 mM 6-aminocaproic acid, 1 mM Phenylmethylsulfonyl fluoride and 0.5 mg/ml type I collagenase at 37 °C for 12–18 h. For the lysis of hypoxia/anoxia treated and control cells, the lysis buffer was supplemented also with 150 µM phloroglucinol to inhibit PXDN activity during the lysis process.
3
Western Blotting for DUOX1 and Kcnq4
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Laemmli sample buffer was added to the cell lysate samples and these were run on 8% or 10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were blocked in phosphate buffered saline containing 0,1% Tween- 20 and 5% dry milk. The first antibodies were diluted in blocking buffer and used either for 1 h at room temperature or overnight at 4 °C. After several washing steps in PBS-Tween-20 membranes were incubated with HRP-linked secondary antibodies (Amersham Pharmaceuticals, Amersham, UK) diluted in blocking buffer. After further PBS-Tween-20 washing steps antibody binding was detected using enhanced chemiluminescence and Fuji Super RX medical X-ray films. Importantly samples were never boiled when processed for western blotting with the DUOX1 antibody.
The redox state of Kcnq4 was also measured by immunoblotting. Detection of proteins was performed using HRP-Conjugated Streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) in phosphate buffered saline containing 0,1% Tween- 20 and 5% bovine serum albumin.
The redox state of Kcnq4 was also measured by immunoblotting. Detection of proteins was performed using HRP-Conjugated Streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) in phosphate buffered saline containing 0,1% Tween- 20 and 5% bovine serum albumin.
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