A29L protein and its related polypeptides were separated using 12% SDS-PAGE separation and then transferred to 0.22-μm nitrocellulose membranes (GE Healthcare). The polypeptides were synthesized by GenScript Biotech Corporation and the terminal group of each peptide is linked to the primary amine on KLH by a crosslinker (crosslinker). The blotted membranes were blocked with 5% skim milk in tris-buffered saline and then incubated with the antibody at 37°C for 60 min. After washing thrice with PBST, the membranes were incubated with an HRP-conjugated goat anti-mouse antibody at 37°C for 60 min and visualized using chemiluminescent HRP substrates.
0.22 μm nitrocellulose membrane
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The 0.22-μm nitrocellulose membrane is a laboratory filtration material used for various applications. It has a pore size of 0.22 micrometers, which allows for the effective separation and retention of particles, microorganisms, and other small components from liquids.
Automatically generated - may contain errors
Lab products found in correlation
Semi dry transfer cell, by Bio-Rad (3 mentions)
Enhanced chemiluminescence luminol reagent, by GE Healthcare (2 mentions)
Ecl blotting detection kit, by Bio-Rad (2 mentions)
Las 4000 mini luminescent image analyzer, by Fujifilm (2 mentions)
Ecl substrate, by Promega (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Molico, by Nestlé (1 mentions)
Nf κb p65 phospho s536, by Abcam (1 mentions)
Anti gapdh clone 71.1, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Rabbit anti beta actin, by Merck Group (1 mentions)
Goat anti il 1β p17 subunit, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Ultrapure lps, by Merck Group (1 mentions)
Ab181547, by Abcam (1 mentions)
Secondary anti goat igg antibody, by R&D Systems (1 mentions)
9 protocols using 0.22 μm nitrocellulose membrane
1
Separation and Visualization of A29L Protein
2
BMDM IL-1β Secretion Assay
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A total of 4 ×106 BMDMs were seeded per well in 6-well plates, infected with NcLiv tachyzoites for 18 h. The supernatants were collected and cells were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, and 0.1% SDS) supplemented with protease inhibitors cocktail (Roche). Lysates and supernatants were boiled in Laemmli buffer, resolved by SDS-PAGE 12%, and transferred (Semidry Transfer Cell, Bio-Rad) to 0.22-μm nitrocellulose membranes (GE Healthcare). The rabbit anti–IL-1β/IL-1F2 polyclonal antibodies (Novus; 1:1000), and species-specific horseradish peroxidase-conjugated secondary antibodies (R&D Systems; 1:1000) were used for antigen detection. The blot was incubated with ECL substrate (Promega) and chemiluminescence was detected using an imaging system with dedicated software (ChemiDoc XRS, Bio-Rad).
3
Quantitative NF-κB Signaling Pathway Analysis
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After removing the supernatants, J774.1 cells were lysed in radioimmunoprecipitation assay buffer RIPA buffer (Merk, Darmstadt, Germany) containing protease and phosphatase inhibitors. Protein quantification was performed using detergent-compatible methodology (DC Protein Assay, Bio-rad, CA, United States). Proteins were separated by polyacrylamide gel electrophoresis (Bolt Bis-Tris 4–12% Plus Gel, Life Technologies, CA, United States) and transferred to 0.22 μm nitrocellulose membrane (GE Healthcare, Madison, WI, United States). The membranes were blocked in Tris buffered saline (TBS) solution containing 0.01% Tween, and 5% non-fat dry milk (Molico, Nestlé, Bebey, Switzerland). Recombinant anti-NF-κB p65 antibody [E379] (Abcam, United States) and Phospho-NF-κB p65 (S536; Abcam, United States) were added at 1:5000 dilution in blocking solution; Anti-GAPDH clone (71.1; Sigma-Aldrich, St. Louis, MO, United States) was added at 1:20,000 dilution. HRP-conjugated antibodies (KPL, Gaithersburg, MD, United States) were used at the dilution of 1:5000. ECL (GE Healthcare, Chicago, IL, United States) was used for band detection. Quantification was performed using Software ImageJ 1.52a (NIH, MD, United States). Data are representative of arbitrary units relative to the control (GAPDH).
4
Immunoblotting analysis of inflammasome components
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BMDMs were lysed with a RIPA buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% (w/v) sodium deoxycholate and 0.1% (w/v) SDS) supplemented with a protease inhibitor cocktail. Precleared lysates and supernatants were boiled with Laemmli buffer, resolved by SDS–polyacrylamide gel electrophoresis and transferred to a 0.22-μm nitrocellulose membrane (GE Healthcare). Rat anti-caspase-1 p20 monoclonal antibody clone 4B4 (Genentech, 1:500), mouse anti-caspase-1 p10 (Santa Cruz Biotechnology, 1:250), goat anti-IL-1β p17 subunit (Sigma-Aldrich, 1:250), rat anti-caspase-11 p10 (Abcam, 1:500) and rabbit anti-beta actin (Sigma-Aldrich, 1:5,000) were used for antigen detection. To detect the expression of Flag-fused effectors by L. pneumophila, bacterial heavy patches streaked from CYE plates after 2 days of growth in the presence of 1 mM IPTG were boiled in Laemmli buffer, immunoblotting was carried out as described above, and anti-Flag M2 (Sigma-Aldrich, 1:5,000) antibody was used for antigen detection.
5
MAYV Infection of Macrophages
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A total of 107 BMDMs were seeded in 6-well plates overnight and then primed with 300 ng/ml PAM(3)CSK(4) (InvivoGen, tlrl-pms) for 4 hours prior to infection with MAYV or mock infection. After 24 hours the supernatants were harvested and proteins were precipitated with ice-cold 50% trichloroacetic acid followed by acetone. Cells were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate and 0.1% SDS) in the presence of a protease inhibitor cocktail (complete, Roche). Precleared lysates and supernatants were boiled in Laemmli sample buffer, resolved by SDS-PAGE and transferred (Semidry Transfer Cell, Bio-Rad) to a 0.22-μm nitrocellulose membrane (GE Healthcare). The membranes were blocked in Tris-buffered saline (TBS) with 0.01% Tween-20 and 5% nonfat dry milk. Rat monoclonal antibody to CASP1 p20 (1:250, Genentech, 4B4), goat antibody to IL-1β p-17 (1:200, Sigma Aldrich, I3767), mouse anti-β-Actin (1:1000, C4, Santa Cruz sc-47778) and specific horseradish peroxidase–conjugated antibodies (1:3,000, KPL, 14-16-06 and 14-13-06) were diluted in blocking buffer for the incubations. Enhanced chemiluminescence luminol reagent (GE Healthcare) was used for antibody detection.
6
Inflammasome Activation Assay in BMDMs
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BMDMs from C57BL/6 (WT), Nlrp3−/−, Asc−/− and Casp1/11−/− mice were seeded at 106 per well, pre-treated with ultrapure LPS (1,000 ng ml−1; Sigma-Aldrich) for 4 h and then either incubated with TsV (50 μg ml−1) for 24 h or stimulated with 20 μM nigericin28 (link) (Sigma-Aldrich) for 1 h at 37 °C (5% CO2). The supernatants were collected and suspended with the Laemmli buffer, boiled, resolved by SDS–polyacrylamide gel electrophoresis (15% gel) and transferred (Semidry Transfer Cell, Bio-Rad, Hercules, CA, USA) onto a 0.22-μm nitrocellulose membrane (GE Healthcare, Madison, WI, USA). The membranes were blocked with Tris-buffered saline containing 0.01% Tween 20 and 5% non-fat dry milk. The rat monoclonal antibody to caspase 1 (p20) clone 4B4 (Genentech) and specific horseradish peroxidase-conjugated antibodies (1:3,000; KPL, Gaithersburg, MD, USA) were diluted in blocking buffer for the incubation. The Enhanced Chemiluminescence Luminol Reagent (GE Healthcare) was used for antibody detection. Densitometric analyses were performed by using the Image J 1.34 s Software (National Institutes of Health, Bethesda, MD, USA). The quantitative densitometric data were expressed as a percentage of an increase relative to baseline control levels.
7
Insulin Oligomer Detection Protocol
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Insulin samples (2 μL) were spotted onto a nitrocellulose membrane (0.22 μm, GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was blocked with 5% skim milk in 5% BSA/Tris-buffered saline (TBS) containing 0.01% Tween 20 for 1 h at room temperature. Subsequently, the membrane was incubated with primary anti-insulin antibody (1:3000, ab181547, Abcam, Cambridge, MA, USA) and anti-oligomer antibody (1:3000, A11, Invitrogen, Carlsbad, CA, USA) for 24 h. The following day, the membrane was incubated with the secondary anti-rabbit IgG antibody (1:10,000; R&D Systems, Minneapolis, MN, USA) for 1 h, as described10 (link). After washing with TBST, proteins were visualized using an ECL blotting detection kit (Bio-Rad) according to the manufacturer’s instructions. Luminescence was detected with a Las 4000 mini luminescent image analyzer (Fujifilm, Tokyo, Japan) using the Image Reader Las 4000 software. ImageJ was used to determine the intensity of each dot, which was used to quantify the amount of antibodies on the membrane.
8
Quantitative Insulin Immunodetection Protocol
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Insulin sample (2 μL) was spotted onto a nitrocellulose membrane (0.22 μm, GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.01% Tween 20 for 1 h at room temperature. Subsequently, the membrane was incubated with primary insulin antibody (1:2000, Santa Cruz Biotechnology, Texas, US) for 24 h and incubated with the secondary anti-goat IgG antibody (1:10,000, R&D Systems, Minneapolis, MN, USA) for 1 h. Proteins were visualized using the ECL blotting detection kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Luminescence was detected with a Las 4000 mini luminescent Image Analyzer (Fujifilm, Tokyo, Japan) using the Image Reader Las 4000 software. ImageJ was used to determine the intensity of each dot, which was used to quantify the amount of antibody on the membrane.
9
Slot Blot Analysis of Protein Aggregation
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Protein samples from the aggregation assays were diluted in Tris-buffered saline (TBS) 1% (v/v) SDS and loaded onto a pre-equilibrated nitrocellulose membrane (0.22 μm) (GE Healthcare, Chicago, IL, USA) in a slot blot apparatus. Samples were allowed to pass through the membrane by vacuum, and the slots were washed twice with TBS 1% (v/v) SDS. Immunoblotting was performed following standard procedures using anti-IAPP antibody. Images were acquired using an Odyssey® Fc Imaging System (LI-COR Biosciences) and Image Studio® software.
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