Cells (3 × 105/well) were seeded on six-well plates, incubated for 24 h, and lysed. Denatured proteins (20 µg) were separated on 4–12% Tricine gels (Invitrogen), electroblotted onto PVDF membranes, and probed with primary (1:1000 dilution) and LI-COR infrared-based secondary antibodies (1:5000 dilution). Protein bands were quantified by densitometry using an LI-COR image scanner. A complete list of antibodies is provided in Supplementary Table S2 .
Image scanner
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Sourced in United States
The Image Scanner is a high-resolution scanning device used to capture digital images of physical documents, photographs, or other visual materials. It converts analog visual information into a digital format that can be stored, edited, and shared electronically.
Automatically generated - may contain errors
Lab products found in correlation
Tricine gel, by Thermo Fisher Scientific (1 mentions)
Semi dry electrophoretic transfer system, by Bio-Rad (1 mentions)
Dye binding protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Irdye 800cw goat anti mouse antibody, by LI COR (1 mentions)
Dylight 800, by Thermo Fisher Scientific (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Phosphostop, by Roche (1 mentions)
4 protocols using image scanner
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Protein Analysis
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Cells were lysed with RIPA buffer (50 mM Tris-HCL, pH 7.4, 50 mM NaCl, 2 mM EDTA, 0.1% SDS plus freshly added proteinase inhibitor cocktail (including apoprotein, leupeptin, DTT and PMSF)). Protein concentrations in the supernatant were determined using a protein dye-binding assay (Bio-Rad). Total proteins were then subjected to immunoblot analysis. Briefly, after heating for 5 min at 95 °C followed by cooling on ice, 10 µg of total protein was mixed with 2X sample loading buffer (0.5 M Tris-HCL, pH 6.8, 10% SDS, 0.1% bromophenol blue, 20% glycerol, 2% ß-mercaptoethanol) and loaded into each lane before performing electrophoresis with a Mini-protein 3 Cell System (Bio-Rad). The proteins were transferred to a PVDF membrane (Bio-Rad) in transfer buffer (20 mM Tris, pH 8.0, 150 mM glycine, 20% methanol) with a semi-dry electrophoretic transfer system (Bio-Rad). The membrane was then blocked with blocking buffer (Li-COR, Lincoln, USA) at room temperature for 1 h, and then exposed to primary antibodies (Santa Cruz Biotechnology) at 4 °C overnight. Targeted proteins were subsequently detected using IRDye 800CW goat anti-mouse antibody (Li-COR, Lincoln, USA) for 1 h at room temperature and dark. Bands were visualized with an image scanner (Li-COR, Lincoln, USA).
3
Western Blotting of LysA Protein
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Western blotting assay was carried out essentially as described previously (23 (link)). Anti-gp10 antibodies were used as primary antibodies for probing LysA protein and its variant. Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 800 (catalog no. SA5-35571; Invitrogen) was used as secondary antibody. The blot was imaged on LI-COR image scanner.
4
Western Blot Analysis of Inflammatory Response
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Scr, ZIP, and OE MIN6 cells were cultured in a 12-well plate at a seeding density of 4×105 cells/well. Once the cells reached ~75–80% confluency, they were treated with pro-inflammatory cytokines for 24 hours. After treatment, the cells were washed with PBS and lysed with lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 0.05% Deoxycholate, 0.1% IGEPAL, 0.1% SDS, 0.2% x Sarcosyl, 5% Glycerol, 1mM DTT, 1 mM EDTA, 2 mM MgCL2, Protease inhibitor (04693132001; Roche, Complete mini-EDTA free), and phosphatase inhibitor (04906837001; Roche, PhosphoStop). Protein concentrations were determined using Lowry’s method. Twenty micrograms of protein per sample was heated for 10 minutes at 70 °C and immediately placed on ice. Proteins were separated using a 4–12% Bis-Tris plus gel (NW04122BOX; Invitrogen) and transferred to a PVDF membrane. Membranes were blocked with Intercept blocking buffer (927–70001; LI-COR) and incubated overnight at 4 °C with primary antibodies against β-tubulin (2128; Cell Signaling), caspase-3 (9662; Cell Signaling), and cleaved caspase-3 (9664; Cell Signaling). Membranes were then washed with 0.05% phosphate buffered saline-Tween (PBS-T) for 20 minutes and incubated with donkey anti-rabbit and donkey anti-goat secondary antibodies (LI-COR) for 2 hours. Images were acquired using an LI-COR image scanner, and protein was quantified using ImageStudio software.
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