Changes in TJ protein expression were quantified by Western blot analysis. Cells were washed twice with ice-cold phosphate-buffered saline (Gibco, Life Technologies) and lysed with whole cell lysis buffer containing 150 mM NaCl, 10 mM Tris buffer (pH 7.5), 0.5% Triton X-100, 1% SDS, and Complete Protease Inhibitor (Roche AG, Manheim, Germany). Lysed cells were scraped from the filters, transferred to reaction tubes, and incubated for 30 min on ice with vortexing in between and centrifuged afterward for 30 min at 15,000 × g at 4°C. Proteins were loaded on 12.5% polyacrylamide gels. Primary antibodies claudin-1 (1:1000; Invitrogen, Carlsbad CA, USA), claudin-3 (1:1000; Invitrogen), claudin-4 (1:1000; Invitrogen), and, as loading control, β-actin (1:10,000; Sigma Aldrich) were incubated over night at 4°C. The following day, peroxidase-conjugated secondary antibody, either goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch, Ely, UK), was incubated for 2 h at room temperature. Detection of proteins was performed by membrane incubation with SuperSignal West Pico PLUS Stable Peroxide Solution (Thermo Scientific, Waltham, MA, USA). Visualization of proteins was by the Fusion FX imaging system and Fusion FX6 Edge software (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany), and densitometry was performed using ImageJ software.
Supersignal west pico plus stable peroxide solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Pico PLUS Stable Peroxide Solution is a chemiluminescent substrate designed for the detection of horseradish peroxidase (HRP) in Western blotting applications. The solution is formulated to provide a stable, consistent signal over an extended period of time.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (3 mentions)
Claudin 4, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Claudin 1, by Thermo Fisher Scientific (2 mentions)
Claudin 3, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Enhancer solution, by Thermo Fisher Scientific (1 mentions)
Bp377 500, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti rabbit igg hrp linked antibody 7074s, by Cell Signaling Technology (1 mentions)
Super signal west pico plus luminol enhancer solution, by Thermo Fisher Scientific (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Fusion fx7 imaging system, by Vilber (1 mentions)
Occludin, by Merck Group (1 mentions)
7 protocols using supersignal west pico plus stable peroxide solution
1
Quantifying TJ Protein Expression
2
Immunoprecipitation Protein Extraction and Western Blot Analysis
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Total protein (20–30 µg) was extracted using immunoprecipitation lysis buffer (3% NaCl, 5% Tris–HCl, 10% Glycerol, 0.5% Triton X-10 in Milli Q water) and resolved on 10% SDS-PAGE gels followed by western blotting on Immuno-Blot PVDF membranes (Bio-Rad). Antibodies used were diluted in 5% milk. Membranes were washed with TBST containing 10% TBS and 0.1% Tween 20 (Fisher Chemicals BP377-500). Images were developed by Super Signal West Pico PLUS Stable Peroxide Solution (Thermo Scientific 1863095) and Enhancer Solution (Thermo Scientific CN: 1863094) using a chemiluminescence imager (GE Bio-Sciences AB Amersham Imager 680). Primary and secondary antibodies used are described in Supplemental Table 3.
3
Western Blot Analysis of FGF-10 Isoforms
4
Quantifying TJ Protein Expression
5
Western Blot Analysis of Tight Junction Proteins
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For Western blotting analysis, the protocol was followed as previously described [9 (link)]. The primary antibodies used were occludin (1:1000; Sigma Aldrich, St. Louis, MO, USA), claudin-5 (1:1000; Invitrogen, Carlsbad, CA, USA) and actin (1:1000; Sigma Aldrich, St. Louis, MO, USA). The peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch, Ely, UK). SuperSignal West Pico PLUS Stable Peroxide Solution (Thermo Scientific, Waltham, MA, USA) was used for protein detection, and Fusion FX7 imaging system (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) was used to detect protein signal levels.
6
Western Blotting and Co-Immunoprecipitation Protocols
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Harvested cells were washed with PBS and lysed with ice-cold NETN buffer [20 mM Tris·HCl (pH 8.0), 100 mM NaCl, 0.5% Nonidet P40, and 1 mM EDTA] with the addition of Benzonase nuclease (ChemCruz) for 30 min on ice. Whole cell lysates were subsequently subjected to SDS-PAGE and transferred onto PVDF membrane. After blocking in 5% milk for 1h, the membranes were incubated with primary antibodies diluted in 3% bovine serum albumin (BSA)/Tris-buffered saline + Tween 20 (TBST) at 4°C overnight followed by incubation with peroxidase-coupled secondary antibodies at room temperature (RT). Proteins were detected by chemiluminescence solutions (SuperSignal West Pico PLUS Stable Peroxide Solution and Luminol/Enhancer Solution, Thermo Scientific) using ChemiDoc MP Imaging System (Bio-Rad). For co-immunoprecipitation, whole cell extracts were centrifuged at 15 000 rpm for 15 min at 4°C after lysis, supernatants were incubated with either streptavidin-conjugated beads (GE Healthcare, Sigma-Aldrich) for 4 h or Protein A beads overnight at 4°C. The beads were washed with ice-cold NETN buffer three times prior to immunoblotting.
7
Quantitative Western Blot Analysis
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Western blotting was performed as previously described [27 ]. 20 μg of the obtained protein were electrophoresed using 12.5% sodium dodecyl sulphate–polyacrylamide gel and then transferred to a PVDF membrane (Perkin Elmer, Rodgau, Germany). After blocking with 1% polyvinylpyrrolidone-40 and 0.05% Tween-20, corresponding protein was detected by primary antibodies against tricellulin (1:2000, Invitrogen), angulin-1 (1:3000, Sigma), claudin-4 (1:1000, Invitrogen) and β-actin (1:10,000, Invitrogen) and secondary peroxidase-conjugated antibodies (anti-mouse or anti-rabbit, Jackson ImmunoResearch, Ely, UK). For detection, membranes were incubated with SuperSignal West Pico Plus Stable Peroxide solution (Thermo Fisher, MA, USA) and exposed in Fusion FX7 (Vilber Lourmat, Eberhardzell, Germany). Densitometric analysis was performed using Multi Gauge V2.3 software (FujiFilm, Japan). The protein expression was quantified after normalizing the respective band intensities by β-actin.
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