Lung tissues were homogenized in RIPA lysis buffer containing phosphatase inhibitors (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Two milligrams of each lysate were applied to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-P membranes (GE Healthcare Ltd, Buckinghamshire, UK). After blocking with Blocking One-P (Nacalai Tesque Inc, Kyoto, Japan), the membranes were incubated with anti-AQP5 (GTX11586, x2000, GeneTex, Inc., Irvine, CA, USA) or anti-β-actin (PM053, 1:1000, MBL Co., Nagoya, Japan). They were washed and incubated with peroxidase-conjugated anti-rabbit IgG (1:5000, MBL Co.). The protein bands were visualized with Chemi-Lumi reagents (Nacalai Tesque Inc) and image-captured with a CCD camera system, ImageQuant LAS 4000 mini (GE Healthcare Ltd).
Hybond p membrane
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Spain, Germany, Japan
The Hybond-P membrane is a polyvinylidene fluoride (PVDF) transfer membrane used in Western blotting applications. It provides a support medium for the immobilization of proteins separated by gel electrophoresis, enabling their subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Western blot ecl plus kit, by GE Healthcare (5 mentions)
Ecl western blotting system, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Westsave, by AbFrontier (4 mentions)
X ray film, by MidSci (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
β actin, by Merck Group (3 mentions)
Typhoon trio, by GE Healthcare (3 mentions)
Anti mouse igg hrp conjugate, by Promega (3 mentions)
Image quant 300 cabinet, by GE Healthcare (3 mentions)
Anti nos2, by Santa Cruz Biotechnology (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Trans blot cell, by Bio-Rad (3 mentions)
Phosphatase inhibitor, by Santa Cruz Biotechnology (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Chemi lumi reagents, by Nacalai Tesque (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Ecl plus detection system, by GE Healthcare (2 mentions)
Smad2 3, by Cell Signaling Technology (2 mentions)
108 protocols using hybond p membrane
1
Quantification of Aquaporin-5 in Lung Tissue
2
Western Blot Analysis of Mouse Tissue Proteins
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We homogenized mouse tissues in buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 1 mM 2‐mercaptoethanol with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, NJ, USA) and then centrifuged them at 2,500 × g for 15 min. Equal amounts of protein were separated by 5–20% SDS–PAGE and transferred to Hybond‐P membranes (GE Healthcare, Piscataway, NJ, USA). The primary antibodies and their dilutions were as follows: AR (N20, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Dnmt1 (1:1,000; Abcam, Cambridge, MA, USA), Dnmt3a (1:1,000; Abcam), Dnmt3b (1:1,000; Abcam), Hes5 (1:1,000; Santa Cruz), and ChAT (1:1,000; Millipore, Billerica, MA, USA). Primary antibody binding was probed with horseradish peroxidase‐conjugated secondary antibodies at a dilution of 1:5,000, and bands were detected by using an immunoreaction enhancing solution (Can Get Signal; Toyobo, Osaka, Japan) and enhanced chemiluminescence (ECL Prime; GE Healthcare). An LAS‐3000 imaging system (Fujifilm, Tokyo, Japan) was used to produce digital images. The signal intensities of these independent blots were quantified using IMAGE GAUGE software version 4.22 (Fuji) and expressed in arbitrary units (n = 3 for each group). The membranes were reprobed, or the same samples were examined with an anti‐GAPDH (1:5,000; Santa Cruz) antibody for normalization.
3
Western Blot Analysis of Protein Expression
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Proteins were isolated using a modified RIPA lysis buffer containing 50 mM Tris HCl, 1% NP-40, 1% natrium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4 and 1x Protease inhibitor cocktail (Roche). Equal amounts of protein were electrophoresed through 4–12% Bis/Tris gels (Life Technologies), transferred to Hybond-P membranes (GE Healthcare Biosciences, Pittsburgh, PA, USA), and immunodetected with rat anti-E2A (clone# 826927, clone R&D Systems, Minneappolis, MN), or rabbit anti-GAPDH (Cat. # G9545, Sigma) antibodies. Bands were detected by chemiluminescence using ECL Plus Western Blotting Detection System and HyperFilm (GE Healthcare Biosciences). Quantification by densitometry was performed using Image J Software [27 (link)].
4
Whole-cell Protein Lysate Preparation and Analysis
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Whole-cell protein lysates were prepared from cell line monolayers according to standard protocols [23 (link)]. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany). Proteins were separated by SDS/PAGE as described by Laemmli and colleagues [24 (link)] and transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, UK). Changes in protein expression and phosphorylation as visualized by chemiluminescence (ECL chemi-luminescent reagent, GE Healthcare) were captured and quantified using a FUJI LAS3000 system with Science Lab 2001 ImageGauge 4.0 software (Fujifilm Medical Systems, Stamford, CT).
5
Immunoblotting for Intracellular Protein Levels
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To visualize intracellular protein levels by immunoblotting, whole cell extracts were separated by SDS-PAGE (15% polyacrylamide for RppH-FH and DapF-FLAG, 10% polyacrylamide for RNase E) and wet-transferred onto Hybond-P membranes (GE Healthcare). Proteins of interest were detected with monoclonal mouse anti-His antibodies (Clontech; for RppH-FH) or anti-FLAG M2 antibodies (Sigma; for DapF-FLAG) used at a dilution of 1:5000 or with polyclonal rabbit anti-RNase E antibodies (a gift from George Mackie, University of British Columbia) used at a dilution of 1:100 000. Bands were visualized with goat horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibodies (BioRad), used at a dilution of 1:100 000, in combination with the Supersignal West Pico Maximum Sensitivity Substrate (ThermoFisher Scientific).
6
Bone Marrow-Derived Macrophage Protein Analysis
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BMDMs were lysed in extraction buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 20 μM aprotinin, 0.5% Triton X-100, 50 nM Calyculin A) and incubated on ice for 30 min. Cell extracts were resolved using SDS-PAGE and transferred to Hybond-P membranes (GE Healthcare, Chicago, IL, USA). The membranes were immunoblotted with the indicated antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies using the ECL (GE Healthcare) or SuperSignal West Femto (ThermoFisher Scientific).
7
SDS-PAGE and Immunoblotting Protocol
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Samples were separated by SDS-PAGE and transferred onto Hybond-P membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were incubated with primary antibodies. The antibodies used in the experiments are shown in Supplementary Table 3 (online only).
8
Western Blot for MASH-1 Protein Expression
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Cells were washed in cold PBS, scraped on ice and lyzed in ice-cold lysis buffer (0.5% sodium deoxycholate, 20 mM Tris–HCl, pH 7.4, 0.1 M NaCl, 1% Nonidet P40, 5 mM MgCl2, 1 mM DTT, with protease inhibitors) for 20 min on ice. Samples were separated by SDS-PAGE, transferred to Hybond-P membranes (GE Healthcare, Chicago, IL, Unites States), probed with specific primary and horseradish peroxidase-conjugated secondary Abs, then binding visualized by chemiluminescence (GE Healthcare) with Chemidoc XRS System (Bio-RaD, Hercules, CA, United States). Western Blot analysis of MASH-1 expression was also performed in the presence of K252a, a pan Trk inhibitor (Sigma-Aldrich), [37 (link)]. Densitometry was performed using Imager Chemidoc XRS System.
9
Western Blot Protein Analysis
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Whole cell extracts, cytoplasmic extracts, and nuclear extracts were boiled in Laemmli loading buffer [58.3 mM Tris-HCl (pH 6.8), 1.7% SDS (w/v), 6% glycerol (v/v), and 0.83% 2-mercaptoethanol] and separated on 10–12.5% SDS-polyacrylamide gel electrophoresis. Then the proteins were transferred to 200 mA for 1 h onto Hybond-P membranes (GE Healthcare). Nonspecific binding was blocked for 30 min at room temperature with TBST buffer [20 mM Tris-HCl (pH 7.6), 135 mM NaCl, and 0.1% Tween 20 (v/v)] containing 3% (w/v) nonfat milk. After a short washing in TBST, the membrane was incubated in a 1:1,000 dilution of either primary antibody overnight at 4°C. Primary antibodies were diluted in 3% nonfat milk. In the case when anti-p65 NF-κB antibody was used, it was diluted in solution 1 (Toyobo, Osaka, Japan). After washing for 10–30 min in TBST, the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG (diluted at 1:5,000 in TBST containing 3% nonfat milk) using the ECL Western blotting system (GE Healthcare).
10
Western Blot Analysis of Signaling Proteins
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For Western Blot analysis, cells were lysed in 1× Cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) in either normoxia or hypoxia. Lysis buffer was supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates were then separated on a 8% polyacrylamide gel, transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, United Kingdom), probed with the appropriate antibodies FLT3 (c-20), EGF-R (1005), Epo-R (M-20) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-KIT, p-STAT5, p-AKT (Ser473), GAPDH (all from Cell Signaling Technology, Danvers, MA, USA), β-actin (Sigma-Aldrich, St Louis, MO, USA) and visualized using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). Relative quantification of protein bands from western blot films was performed by using ImageJ software and calculated as optical density (OD). OD was calculated as a ratio of each protein band relative to the lane’s loading control.
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