Tissues were lysed in the lysis buffer for 30 min on ice with vortexing every 5 min. The lysates were then centrifuged at 14,000 rpm for 5 min to remove the insoluble material. Protein concentrations were determined by the Bradford method (Bio-Rad) using BSA as the standard. For Cld-5, the protein was separated on 16% SDS-PAGE gels. The gels were subsequently transferred onto PVDF membranes (Amersham Hybond TM-P, GE Healthcare, Buckinghamshire, UK) by electroblotting for 2 h at 60–75 V. The membranes were then blocked with 5% nonfat milk solution in Tris-NaCl buffer (TNT) containing 0.5% Tween-20 and incubated with primary antibodies as indicated. Monoclonal donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies were used at 1 : 3,000. Proteins were detected by enhanced chemiluminescence using a commercial kit (Amersham Hybond TM-P, GE Healthcare, Buckinghamshire, UK).
Amersham hybond p
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Amersham Hybond-P is a porous polyvinylidene difluoride (PVDF) membrane designed for protein transfer and immobilization in western blotting applications. It provides high protein binding capacity and ensures efficient transfer of proteins from polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (63 mentions)
Ecl plus western blotting detection system, by GE Healthcare (13 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
Amersham ecl prime, by Cytiva (4 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
Las 4000, by Cytiva (4 mentions)
Ecl plus kit, by Cytiva (4 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Antibody against gapdh, by Abcam (3 mentions)
Zr bashingbead beads, by Zymo Research (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Nupage 10 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Ecl prime blocking agent, by GE Healthcare (2 mentions)
Las 3000 imager, by Fujifilm (2 mentions)
Hrp conjugated rabbit anti human igg, by Merck Group (2 mentions)
Anti his mouse monoclonal primary antibody, by Qiagen (2 mentions)
Imagequant las 4000 developer, by GE Healthcare (2 mentions)
72 protocols using amersham hybond p
1
Western Blot Analysis of Claudin-5
2
Protein Extraction and Analysis
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To prepare lysates, cells were incubated for the indicated time and concentration of Pt-1-DMCa, collected after trypsinization and washed twice with cold PBS. Total cell lysates were scrapped in ice-cold lysis buffer (10 mM Tris pH 7.5, 0.5 mM EDTA pH 8.0, 0.5 mM DTT, 0.5% CHAPS, 10% glycerol) supplemented with a cocktail of protease inhibitors. Specifically, nuclear fractions were obtained using the NE-PER nuclear/cytoplasmic extraction kit. Mitochondria were fractionated using the Mitochondria Isolation Kit for Mammalian Cells (Thermo Fisher Scientific, Rockford, IL, USA). Proteins in each sample were determined using the DC Protein Assay (Bio-Rad) according to the manufacturer's specifications. After normalization, equal amounts of proteins were fractionated on 8-15% SDS-PAGE gels. The proteins were then transferred overnight or 2 hrs at 4°C to a polyvinylidene difluoride (PVDF) membrane (Amersham Hybond™-P, GE Healthcare, Buckinghamshire, UK) and incubated with the indicated primary antibodies and corresponding HRP conjugated secondary antibodies. The immunoreactive bands were visualized by chemiluminescence according to the manufacturer's recommendations (ECL, Amersham Pharmacia (Amersham Hybond™-P, GE Healthcare, Buckinghamshire, UK). The ECL staining was quantified by densitometry with ImageJ software (National Institutes of Health, USA).
3
Western Blot Analysis of Cellular Proteins
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We denatured tissue lysate proteins (40 µg) by heating at 100 °C for 5 minutes; separeted the proteins using SDS-PAGE and transferred to PVDF membranes (Amersham HybondTM–P, GE Healthcare). We blocked the blotted membranes with 5% bovine serum albumin overnight at 4 °C. We detected individual proteins by blotting with specific primary antibodies for 1 hour at room temperature using anti-VDAC (ab15895, 1:1000, Abcam), anti-COX IV (#4840, 1:1000, Cell Signaling), anti-CDK1 (ab32384, 1:1000, Abcam), anti-LC3 (#2775, 1:1000, Cell Signaling) and anti-beta-actin (ab8227, 1:2000, Abcam) followed by secondary probing with HRP-conjugated (1:1000 dilution). Immunodetection was determined using the enhanced chemiluminescence (ECL) method. We used ImageJ software (NIH software) for densitometry normalized it with beta-actin expression.
4
Western Blot Analysis of cIAP1 and cIAP2
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For Western blots, 50 μg of the whole cell lysate was resolved on 10% SDS-PAGE gel and transferred to PVDF membranes (Amersham Hybondtm-P, GE Healthcare, USA), followed by Western blotting with antibodies for cIAP1 and cIAP2 (antibody details are provided in Supplementary Table S6 ). The blots were developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, USA), according to the manufacturer’s instructions and quantified with Image J software.
5
Quantifying ACE2 and TMPRSS2 Protein Levels
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After treatment with cannabis extracts for the indicated time, whole cellular lysates of 3D tissues were prepared in radioimmunoprecipitation assay buffer using 2.0 mm ZR BashingBead beads (Zymo Research). tissue lysates were prepared from all samples, then equal amount of each sample from each group were pooled together. Proteins (30-100 μg per sample) were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (Amersham HybondTM-P, GE Healthcare) at 4° C for 1.5 h. The blots were incubated for 1 h with 5% nonfat dry milk to block nonspecific binding sites and subsequently incubated at 4° C overnight with 1:1000 dilution of polyclonal antibody against ACE2 (Abcam) or TMPRSS2 (Abcam). Immunoreactivity was detected using a peroxidase-conjugated antibody and visualized with the ECL Plus Western Blotting Detection System (GE Healthcare). The blots were stripped before reprobing with antibody against GAPDH (Santa Cruz Biotechnology). Quantification of Western blot bands was performed using ImageJ in duplicate or triplicate as technical replicates.
6
Endothelial Cell Protein Expression Analysis
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Total cell lysates were prepared from MoAoECs and HUVECs in radioimmunoprecipitation assay (RIPA) (Thermo Fisher Scientific, Waltham, MA, USA) buffer with cOmplete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor (Sigma-Aldrich). Protein concentration was determined with Micro BCATM Protein Assay Kit (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Total protein lysates (25–30 µg/lane) were prepared in 4× Laemmli buffer supplemented with β-mercaptoethanol and preheated (at 90 °C) for separation in SDS-PAGE 4–20% (Bio-Rad, Hercules, CA, USA). Polyvinylidene difluoride (PVDF) membranes (GE Healthcare Amersham HybondTM-P) were probed with rabbit anti-mouse/human-ICAM-1, rabbit anti-mouse/human-VCAM-1 and rabbit anti-mouse/human-E-selectin (Santa Cruz Biotechnology, Dallas, TX, USA) at dilution of 1:500 overnight. HRP-labeled goat anti-rabbit and goat anti-mouse (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were used at dilution of 1:5000 and incubated at RT for 45 min. Human β-actin HRP-conjugated antibody (Alpha Diagnostic International, San Antonio, TX, USA) and mouse anti-α-tubulin were used as loading controls at dilutions of 1:20,000 and 1:1000 respectively. Proteins of interest were detected by Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK).
7
Cannabis Extracts Modulate Inflammatory Proteins
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After treatment with cannabis extracts for the indicated time, whole cellular lysates of 3D tissues were prepared in radioimmunoprecipitation assay buffer using 2.0 mm ZR BashingBead beads (Zymo Research). Proteins (30-100 μg per sample) were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (Amersham HybondTM-P, GE Healthcare) at 4° C for 1.5 h. The blots were incubated for 1 h with 5% nonfat dry milk to block nonspecific binding sites and subsequently incubated at 4° C overnight with 1:1000 dilution of polyclonal antibody against IL-6 and COX-2 (Abcam). Immunoreactivity was detected using a peroxidase-conjugated antibody and visualized with the ECL Plus Western Blotting Detection System (GE Healthcare). The blots were stripped before reprobing with antibody against actin (Santa Cruz Biotechnology) or GAPDH (Abcam).
8
Extraction and Analysis of PIWIL Proteins
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RASF were trypsinized and nuclear and cytoplasmic extracts prepared with NC-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturers protocol. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Amersham HybondTM P; GE Healthcare). Membranes were blocked with 5% milk in TBS-T and incubated overnight at 4°C with rabbit anti-human PIWIL4 (PA5-31448; Thermo Scientific), rabbit anti-human PIWIL2 (sc-67303; Santa Cruz), rabbit anti-human Lamin B1 (ab16048; Abcam) and mouse anti-human α-tubulin (ab7291; Abcam) antibodies in 5% milk. HRP-labeled species-specific secondary antibodies (Jackson Immuno Research) were used and signals were detected using enhanced chemiluminescence Western BrightTM ECL (Advansta) and the Fusion FX imager (Vilber Lourmat). The Alpha Imager software system (Alpha Innotech) was used to analyse the results.
9
Quantification of TRF2 Protein Expression
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Western blots were performed following Bio-Rad protocol. Briefly, 20 µg proteins were electrophoresed in SDS-PAGE (SDS-Polyacrylamide 10%) under denaturing conditions, then transferred to a PVDF membrane (Polyvinylidin Difluoride) (Amersham Hybond TM P + , GE Healthcare) or nitrocellulose, which were hybridized with mouse monoclonal anti-TRF2 antibody (4A794, Upstate), the Anti-TRF2 Thr-188P polyclonal antibody and anti-actin HRP (SC1616-HRP, Santa-Cruz). TRF2 was revealed by the secondary antibody goat anti-mouse IgG-HRP (ab6789, abcam) using the ECL Western Blotting detection reagent. Western-blot membranes were analysed using FluorChem software program.
10
Western Blot Analysis of Protein Expression
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Forty-eight hours after siRNA transfection, asynchronous cells were lysed in TEN-N buffer (1% NP-40, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). Protein concentration was measured by the Bio-Lad Protein assay kit (Bio-Rad, Hercules, CA, USA). Extracted proteins were boiled for 10 min with 4 × NuPAGE® LDS (lithium dodecyl sulfate) sample buffer (Thermo Fisher Scientific). Proteins were separated by NuPAGE® SDS (sodium dodecyl sulfate) PAGE (polyacrylamide gel electrophoresis)-Gel System (Thermo Fisher Scientific), electroblotted onto a polyvinylidene difluoride membrane (Amersham Hybond-P, GE Healthcare), and reacted with appropriate primary antibodies overnight at 4 °C for immunodetection. Blocking and antibody incubation were performed in 3% non-fat dry milk. Proteins were detected using horseradish peroxidase-labeled secondary antibodies (1:3000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature and enhanced chemiluminescence, according to the manufacturer’s instructions (GE Healthcare).
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