Monoclonal antibodies were absorbed to a nitrocellulose membrane and thenblocking VLPs were bound to the Mab-coated membrane. Following this, VLP binding was determined by specific peroxidase labelled 2D9 Mab binding. In this format, 1 µL per dot containing the Mab dilution was added on the nitrocellulose membrane. The drop was allowed to air dry for 15 min before the blocking solution (5% milk powder dissolved in PBS) was added. All washing steps were carried out in triplicate using PBS-Tween (0.05% PBS-Tw). For peroxidase-labelled antibodies, the Pierce™ ECL Western Blotting Substrate system was used for detection (Thermo Fisher Scientific).
Pierce ecl western blotting substrate system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce™ ECL Western Blotting Substrate system is a chemiluminescent detection reagent used in western blotting assays to visualize and quantify proteins. It is designed to produce a luminescent signal in the presence of the target protein, allowing for the detection and analysis of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (2 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Mcl 1 clone s 19, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Supersignalwest femto maximum sensitivity substrate system, by Thermo Fisher Scientific (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using pierce ecl western blotting substrate system
1
Quantifying VLP Binding to Monoclonal Antibodies
2
Western Blot Analysis of Protein Expression
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RIPA buffer (Sigma-Aldrich, Germany) was used for lysing cells, and total protein was collected by centrifugation at 14,000 × g for 20 min. Protein concentration was determined using a BCA protein assay kit. (Thermo Fisher, USA). Equal amounts of protein lysates were subjected to SDS-PAGE, transferred to methanol-activated PVDF membranes, blocked with 5% nonfat dry milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween (TBST) for 2 h, and incubated at 4°C overnight with primary antibodies (Abcam Biotechnology, UK). Membranes were then incubated with anti-rabbit secondary antibodies (Cell Signaling Technology, China) for 1 h at room temperature. Protein bands were detected using the Pierce ECL Western Blotting Substrate system (Thermo Scientific, Belmont, Massachusetts, US). GAPDH was used as a normalization control for each sample.
3
Western Blot Analysis of Retinal Proteins
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Retinas or retinal EC were lysed in RIPA buffer (Millipore, Billerica, MA, USA) and 30 µg of total protein was separated by SDS-PAGE. Antibodies used are listed in Table 2 . Membranes were visualized using Pierce™ ECL Western Blotting Substrate system (Thermo Fisher Scientific) or Odyssey® CLx Imaging System (LI-COR Biosciences) after incubation with their respective secondary antibodies. Band intensities were quantified using Alpa Innotech Fluorchem (Santa Clara, CA, USA) or image-J imaging and densitometry software and expressed as relative optical densitometry (ROD) compared to the WT-ND control group.
4
Western Blot Analysis of Protein Markers
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Cells were lysed in M-PER buffer (Pierce Biotechnology, Rockford, IL, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Total cell extracts were separated using SDS-PAGE in 10% gels, transferred to nitrocellulose membranes, and blotted as described previously [97 (link)]. The primary antibodies included anti-NGAL (clone AF1757, goat IgG, specific for the dimeric and monomeric forms; R&D Systems), anti-MMP-9 (clone EP1254, rabbit Ig, specific for CPX; Abcam, Paris, France), anti-NGAL-R/Slc22A17 (clone 75010, rabbit Ig; Biorbyt), and antibodies against Mcl-1 (clone S-19, rabbit IgG; Santa-Cruz), Bcl-2 (clone 100, mouse IgG1; Santa-Cruz), STAT3 (clone C20, rabbit IgG; Santa-Cruz), phospho-Tyr705-STAT3 (clone 710093, rabbit Ig; Thermo Fisher Scientific, Waltham, MA, USA), STAT1 (clone -23, rabbit IgG; Santa-Cruz) and actin (clone C4, mouse IgG1; ICN Biomedicals, Solon, OH, USA). Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and visualized with the Pierce ECL Western blotting substrate system or the SuperSignal West Femto Maximum Sensitivity Substrate system (both from Thermo Fisher Scientific). Immunoblot images were acquired in an MF-ChemiBIS 4.2 imager (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel) and quantified using ImageJ64 software.
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