Cells were seeded at a density of 1 × 106 cells/plate on 60 mm cell culture dish overnight. Cells were incubated with M5 or PG102 at designated concentrations for 30 min and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents in the cell lysates were determined by bicinchoninic acid (BCA) assay kit and after reconstituting in sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane (Merck) and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 h to block nonspecific binding. The membrane was then incubated with antibodies specific for phospho-STAT3 (#9134, #9145), STAT1 (#9167), p65 (#3033), and STAT3 (#4904), STAT1 (#9172), p65 (#8242), IκB-α (#9242) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (A5441, Sigma-Aldrich) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich) at room temperature for 1 h. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure on autoradiography film.
Bolt 10 bis tris plus gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bolt 10% Bis-Tris Plus gels are a electrophoresis gel for protein separation. The gels are pre-cast and ready-to-use. They have a Bis-Tris buffer system and a 10% acrylamide concentration.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (5 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (4 mentions)
Iblot2 system, by Thermo Fisher Scientific (4 mentions)
Novex mini cell, by Thermo Fisher Scientific (4 mentions)
Ecl plus western blotting detection system, by GE Healthcare (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
β actin, by Merck Group (3 mentions)
Complete ultra protease inhibitor, by Roche (3 mentions)
Phostop phosphatase inhibitor cocktail, by Roche (3 mentions)
Bioruptor 200, by Diagenode (3 mentions)
Immobilon fl pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Horseradish peroxidase conjugated secondary anti mouse or anti rabbit igg, by Merck Group (2 mentions)
Immobilon ecl hrp substrate, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Phosstop, by Roche (2 mentions)
Cytobuster, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (2 mentions)
25 protocols using bolt 10 bis tris plus gel
1
Western Blot Analysis of Signaling Pathways
2
Protein Expression Analysis in HaCaT Cells Treated with PG102
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HaCaT cells were seeded at a density of 4.5 × 105 cells/well onto 6-well plates overnight. Cells were then treated with PG102 at designated concentrations for 48 hours, and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents were measured by the BCA assay kit (Invitrogen), and after reconstitution in a sample buffer, 20 micrograms of protein samples was subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane, and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 hour to block nonspecific binding. The membrane was then incubated with antibodies specific to IL-37, phopho-Smad3 (1 : 1000; Abcam), Smad3, phospho-ERK, ERK (1 : 1000; Cell Signaling Technology, Danvers, MA), and β-actin (1 : 5000; Sigma-Aldrich) overnight at 4°C followed by incubation with a horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1 : 100,000; Sigma-Aldrich) at room temperature for 1 hour. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure to autoradiography film.
3
SDS-PAGE Protein Separation Protocol
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Protein homogenates (100 μg) were mixed with Laemmli sample buffer and β-mercaptoethanol (3% v/v), and incubated for 5 min at 95 °C. After cooling, 10 μg protein was loaded into Bolt 10% Bis-Tris Plus gels (Invitrogen) and electrophoresed for 30 min at 160 V. Gels were then stained with Coomassie Blue for protein visualization.
4
Western Blot Analysis of PCYT2
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Homogenates were made from controls and patient fibroblasts using RIPA buffer (Sigma-Aldrich). The protein concentration of the homogenates was determined using the BCA Protein Assay Kit (reducing agent compatible) (Pierce). Electrophoresis was carried out using Bolt 10% Bis-Tris Plus Gels and NuPAGE® MES SDS Running Buffers (Invitrogen). For each sample, 35 μg of protein was loaded ontp the polyacrylamide gel and electrophoresed for 45 min at 120 V. The proteins were blotted onto nitrocellulose membranes using Mini iBlot® Gel Transfer Stacks Nitrocellulose (Invitrogen). After blocking non-specific binding, the membrane was incubated overnight at 4°C with specific anti-PCYT2 (ab126142, Abcam) followed by a 1-h incubation with a secondary fluorescent-labelled goat anti-rabbit antibody (IRDye 800CW LI-COR). The signal was developed using an Odyssey CLX imaging machine. The membrane was then washed with PBS-Tween 0.1% buffer. The washed membranes were incubated with anti-GAPDH (5174S, Cell Signalling) for an hour followed by incubation with a secondary fluorescent-labelled goat anti-rabbit antibody (IRDye 680CW LI-COR) and visualized as described above.
5
Immunoblotting Assay for STING Signaling Pathway
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Cell lysates were prepared in Cell Extraction Buffer (Invitrogen) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA), 1 mM PMSF, and 200 µM sodium orthovanadate. Approximately 10~15 ug of whole-cell lysate was resolved by SDS PAGE using Bolt 10% Bis-Tris Plus gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to Immobilon-FL PVDF membranes (EMD Millipore) for immunodetection of proteins. After 90 min blocking at RT in LI-COR blocking buffer, the blots were incubated overnight at 4 °C with primary antibodies and 90 min at RT with mouse or rabbit IRDye® secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Blots were then imaged on an Odyssey FC (LI-COR). Immunoblot images were analyzed by Image Studio (Lite Ver 5.2). The primary antibodies used for immunoblotting include anti-TBK1 (3504, Cell Signaling Technology, 1:1000), anti-p-TBK1 (5483, Cell Signaling Technology, 1:1000), anti-IRF3 (Sc-33641 Santa Cruz, 1:500), anti-p-IRF3 (29047, Cell Signaling Technology, 1:1000), anti-STING (13647, Cell Signaling Technology, 1:500), anti-p-STING (50907, Cell Signaling Technology, 1:1000), and anti-GAPDH (97166, Cell Signaling Technology, 1:1000).
6
Immunoblotting of FAK Phosphorylation
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hMSCs were encapsulated and cultured in adhesive peptide-functionalized hydrogels overnight. Cells were washed twice with PBS and lysed by sonication on ice in cell extraction buffer containing protease and phosphatase inhibitors (ThermoFisher). The lysates were cleared by centrifugation at 10,000 rpm for 15 min at 4 °C and the extract was stored at −80 °C until analysis. Protein concentration was determined using a micro BCA kit (Pierce). Equivalent amounts of reduced, boiled (10 min at 70 °C) lysate were loaded on Bolt 10% Bis-Tris Plus gels (ThermoFisher) and subsequently transferred onto PVDF membranes. Membranes were probed with mouse monoclonal antibody against GAPDH (Abcam), mouse monoclonal antibody against FAK and rabbit polyclonal antibody against FAK [pY397] (ThermoFisher) at a 1:1000 dilution in 5% BSA TBS-T solution followed by fluorescent secondary antibodies (Li-Cor). Immunoblots were visualized on a Li-Cor Odyssey imaging system and analyzed using Image Studio Lite (Li-Cor) (Supplementary Fig. 20 ).
7
Western Blot Analysis of Pluripotency Factors
8
NCAM1 Immunoblotting Assay for Kidney Disease
9
Protein Extraction and Western Blotting Procedure
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Cells were lysed in RIPA buffer (Sigma) containing Complete-ULTRA protease-inhibitor and PhoStop phosphatase-inhibitor cocktails (Roche), and sonicated with Bioruptor200 (Diagenode) at high frequency, alternating 30 s on/off for 3 min. SDS-PAGE electrophoresis was performed using Bolt 10% Bis-Tris Plus gels (ThermoFisher) in a Novex MiniCell (ThermoFisher). Protein transfer was performed using the semi-dry iBlot2 system (ThermoFisher) and iBlot Transfer Stacks (ThermoFisher). The following primary antibodies were used: Esrrb (1:1000, mouse mAb, Perseus Proteomics); Klf2 (kind gifts from Huck-Hui Ng; 1:500, rabbit serum, Yeo et al., 2014 (link); and Hitoshi Niwa (1:1000, mouse mAb; Yamane et al., 2018 (link)); Oct4 (1:1000, rabbit mAb, Cell Signaling); P-Y705-Stat3 (1:1000, rabbit mAb, Cell Signaling); αTubulin (1:10000, mouse mAb, Abcam). Detection was achieved using HRP-linked secondary antibodies at 1:10000 against the appropriate species (GE Healthcare) and ECL Plus Western Blotting Detection System (GE Healthcare).
10
Filaggrin Protein Expression in HaCaT Cells
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HaCaT cells were plated in 100mm culture dishes. Twenty-four hours later, the cells were treated with 0.25, 0.5, 1, 2 mg/mL of ACTPER for 72 h. After treatment, these same cells were washed with cold PBS and lysed using a phosphosafe extraction buffer (Novagen, Madison, WI, USA). Total protein contents in the cell lysates were determined by a Bradford assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. After reconstituting in the sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (ThermoFisher, Waltham, MA, USA), and electrophoretically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were reacted with primary antibodies against filament aggregating protein (Filaggrin) (sc-80609, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (A5441, 1:5000, Sigma). Membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000, Sigma) and visualized in films using ECL solution (Millipore, Billerica, MA, USA).
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