Cells were lysed with RIPA buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were separated by SDS–PAGE under reducing conditions, transferred to a nitrocellulose membrane, and analyzed by immunoblotting. MDHC (H-6) (Cat# sc-166879, RRID:AB_10609257) was purchased from Santa Cruz Biotechnology. GPD1 polyclonal antibody (Cat# 27943-1-AP, RRID:AB_2881016) and GPD1L polyclonal antibody (Cat# 17263-1-AP, RRID:AB_2112359) were purchased from Proteintech. Rabbit anti-Ras (G12V) monoclonal antibody (Cat.#: 14412, RRID:AB_2714031) was purchased from Cell Signaling. Goat anti-rabbit secondary antibodies (HRP conjugated) and anti-mouse secondary antibodies were purchased from LI-COR and Santa Cruz Biotechnology, respectively. Immunoblotting for β-tubulin by rabbit anti-β-tubulin antibody (HRP conjugated) (Cat# 5346, RRID:AB_1950376) (Cell Signaling) was used as a loading control. Signal was detected by using WesternSure premium chemiluminescent substrate and the C-Digit Blot Scanner (LI-COR) according to the manufacturer’s instructions.
Westernsure premium chemiluminescent substrate
Manufactured by LI COR
Sourced in United States, Germany
The WesternSure PREMIUM Chemiluminescent Substrate is a laboratory reagent designed for use in Western blot analysis. It is a chemiluminescent substrate that emits light upon interaction with the enzyme-labeled target proteins, allowing for the detection and visualization of these proteins.
Automatically generated - may contain errors
Lab products found in correlation
C digit blot scanner, by LI COR (22 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pierce co immunoprecipitation kit, by Thermo Fisher Scientific (5 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Odyssey fc imaging system, by LI COR (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Protease and phosphatase inhibitor, by Merck Group (3 mentions)
Neuronal protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Avantor (2 mentions)
Non fat dried milk, by Carl Roth (2 mentions)
Secondary anti rabbit igg antibody conjugated to horseradish peroxidase, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Image studio lite, by LI COR (2 mentions)
48 protocols using westernsure premium chemiluminescent substrate
1
Immunoblotting of Protein Lysates
2
Immunoblotting Assay for PMDH2 and HA Tags
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Immunoblotting was done as described74 (link) except that membranes were air dried for 1 h before blocking overnight in 8% (w/v) non-fat dry milk in TBST (Tris-buffered saline with 0.1% [v/v] Tween-20). Primary antibodies used were rabbit anti-PMDH275 (link) (1:5000), rat anti-HA (1:300, Roche clone 3F10, Sigma 11867423001), and mouse anti-HSC70 (1:50,000, Stressgen SPA-817). Horseradish peroxidase-conjugated secondary antibodies (1:5000 goat anti-rat IgG, Invitrogen A10549; 1:5000 goat anti-rabbit IgG, GenScript A00098; or 1:5000 goat anti-mouse IgG, GenScript A00160) were incubated for 2 h before washing in TBST. Antibodies were imaged using WesternSure Premium Chemiluminescent substrate (LI-COR Biosciences) and a LI-COR Odyssey Fc imaging system. Uncropped blots are included in the Source Data file.
3
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, CA, USA). Protein concentration was determined using the Bradford assay (Sigma-Aldrich). Total proteins were separated by electrophoresis using 4–12% NuPAGE Bis-Tris protein gels (Thermo Fisher Scientific, Carlsbad, CA, USA), transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK), and probed using the conditions indicated in Supplementary Table S2 . Blots were revealed by Western Sure Premium Chemiluminescent Substrate LI-COR and by a C-DiGit Blot scanner (LI-COR Biosciences, Lincoln, NE, USA). Densitometric quantifications were normalized relative to beta-Actin signal using Image Studio software (Image Studio Digits Version 4.0.21) (http://www.licor.com , accessed on 2 April 2019).
4
Quantitative Immunoblot Analysis Protocol
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Immunoblot analyses were performed as previously described (18 (link)). The anti-AGO1 (AS09 527; Agrisera), anti-CUL1 (kindly provided by J.C. del Pozo), and anti-RbcL (AS03 037; Agrisera) primary antibodies were used at 1:10,000, 1:3,000 and 1:2,500 dilutions, respectively. WesternSure HRP Goat anti-Rabbit IgG (LI-COR) secondary antibody was used at 1:50,000 dilution. Detection was performed using the WesternSure PREMIUM Chemiluminescent Substrate (LI-COR) and a C-Digit Blot Scanner (LI-COR). The Image Studio Analysis (LI-COR) software was used for band quantification.
5
Western Blot Analysis of Protein Targets
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Cells were re-suspended in a RIPA buffer containing protease inhibitors (Thermo Scientific, catalog numbers: 78425 and 89900). The cell lysates were separated on 4–12% Bis-Tris gels (Life Technologies, catalog number: BG04120) and transferred to PVDF membranes (Life Technologies, catalog number: LC2005). After incubation with primary antibodies against beta-actin (Santa Cruz Biotech), Tau (Millipore), albumin (Abcam), and adiponectin (Abcam) at 4°C overnight, the membranes were incubated with an HRP-conjugated secondary antibody (1:50,000; Li-Cor) for 1 hour at room temperature. They were then incubated with WesternSure Premium Chemiluminescent Substrate (Li-Cor, catalog number: 926-95000) for 5 minutes and visualized using a Li-Cor C-Digit Blot Scanner.
6
Retinal Protein Extraction and Western Blot
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The retinas from mouse eyes were isolated and suspended in T-PER buffer supplemented with protease and phosphatase inhibitors. Protein isolation was achieved by sonicating the samples for 10 min and incubating them for 1 h on a shaker at 4 °C. Denatured proteins from the retina, RPE/choroid, or serum were separated in reducing Laemmli buffer on a 12%, SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 2 h in blocking buffer (5% skim milk in TBST) before being incubated with the appropriate primary antibody in blocking buffer overnight at 4 °C. Following washing, membranes were incubated for 2 h at room temperature in blocking buffer with appropriate HRP secondary antibodies. Following a final washing step, membranes were developed with lumi-light blotting substrate or WesternSure PREMIUM Chemiluminescent Substrate (Licor, Bad Homburg, Germany).
7
MERS-CoV Protein Expression Detection
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Extracts of noninfected Vero cells or Vero cells infected with MERS-CoV strain EMC were generated at Rocky Mountain Laboratories, loaded onto discontinuous 3 and 7.5% SDS gels (Bio-Rad), and transferred onto nitrocellulose with iBlot Transfer Stacks (Invitrogen iBlot; Life Technologies). Lanes were loaded with alternating infected and noninfected extract samples, and a pair were cut for incubation with DC sera (1:800 in blocking solution) after blocking of the membrane in PBS–0.05% Tween 20–5% dry milk blocking solution for 1 h. Membranes were washed three times with PBS–0.05% Tween 20 after a 2-h incubation with serum and then incubated for another 1.5 h with secondary antibody (1:7,000 in blocking solution; anti-llama IgG-horseradish peroxidase conjugate; Bethyl Laboratories). Following three more washes, the membranes were developed with WesternSure premium chemiluminescent substrate (LI-COR, Lincoln, NE) and read on a C-DiGit Blot Scanner (LI-COR).
8
Western blot analysis of XIAP protein
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Total proteins from the cells were extracted using a RIPA Lysis and Extraction Buffer (Thermo Scientific™, Waltham, MA, USA) including Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Scientific™, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The PVDF membranes were incubated with primary antibodies at 4°C overnight, and then further incubated with secondary antibodies for 30 min on the following day. The immunoreactive signals were visualized using the chemiluminescence reagents WesternSure® PREMIUM Chemiluminescent Substrate (LI-COR®, Lincoln, NE, USA). Images were captured with the Odyssey® XF Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The following antibodies were used: XIAP Antibody (Cell Signaling Technology, Danvers, MA, USA), α-Tubulin Antibody (Cell Signaling Technology, Danvers, MA, USA).
9
Immunoblotting of miRNA Targets and p27
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Cells were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris, pH 7.4, 1% TX-100, 0.2% Na deoxycholic acid, and 0.2% SDS, HALT complete tab [Roche]). Proteins were quantified using the Pierce BCA Protein Assay kit (ThermoFisher Scientific) and equal amounts of protein were run on 4–12% Bis-Tris gradient gels (Invitrogen) according to manufacturer’s instructions. Proteins were transferred by semi-dry electrophoresis (BioRad) onto Immobilon-P PVDF (EMD Millipore). Membranes were incubated overnight at 4°C with anti-p27 3688 (Cell Signaling Technologies) or HoxA7 ab70027 (Abcam), washed then incubated with anti-mouse or anti-rabbit HRP conjugated secondary antibodies (GE Healthcare). Membranes were developed on film with WesternSure Premium Chemiluminescent Substrate (LI-COR). The same Immunoblots were reprobed with β-actin clone AC-15 (Sigma) as loading control. Biotinylated miRNA target immunoblots and p27Kip1 immunoblots were repeated on lysates generated from at least two independent experiments each.
10
Western Blotting Analysis of NLRP Proteins
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