Proteins were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris; Invitrogen) and transferred to nitrocellulose membranes using iBlot Gel Transfer Stacks (Invitrogen). Membranes were blocked in 5% skimmed milk in TBST (0.05 M Tris-base, 0.5 M NaCl supplemented with 0.1% Tween-20), and incubated with primary antibodies diluted in blocking buffer overnight. Membranes were washed three times in 0.5% skimmed milk in TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Following a final washing step, proteins were visualized with the ECL plus Western blotting detection system (GE Healthcare) using a Fuji film LAS4000 system. See Supplementary Figs. 10 and 11 for uncropped Western blot images.
Iblot gel transfer stack
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBlot Gel Transfer Stacks are a component used in the iBlot Gel Transfer Device. They facilitate the transfer of proteins from polyacrylamide gels to membranes for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer device, by Thermo Fisher Scientific (6 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (3 mentions)
Iblot system, by Thermo Fisher Scientific (3 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Nupage gel, by Thermo Fisher Scientific (3 mentions)
Phosphatase inhibitor cocktail, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Carl Roth (2 mentions)
Cell recovery solution, by BD (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (2 mentions)
Antibody against gadph, by Abcam (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris, by Thermo Fisher Scientific (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
61 protocols using iblot gel transfer stack
1
Western Blot Analysis of Proteins
2
Western Blot Analysis of Mycobacterial Proteins
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Samples mixed with 2 × Laemmli sample buffer and 1 M dithiothreitol (95 °C, 10 min), and electrophoresed in NuPAGE 4–12% Bis-Tris Pre-cast gels (BioRad; Hercules, CA, USA) for 50 min at 150 V with Precision Plus ProteinTM Dual Color Standard (BioRad; Hercules, CA, USA) were dry-blotted using nitrocellulose membranes iBlot® Gel Transfer Stacks (Invitrogen, Life Technologies; Carlsbad, CA, USA). Membranes were blocked by rolling in 5% skim milk in PBS-0.05% Tween-20 (Amresco; Solon, OH, USA) (RT, 1 h), followed by incubation in primary antibody from 1) pooled or individual (using a Mini-Protean Multiscreen apparatus (Biorad)) TB and non-TB sera at 1:1000; or 2) rabbit polyclonal anti-whole cell lysate (WCL) antibodies (NR-13819) at 1:5000 or pooled mouse monoclonal anti-LpqH (NR-13822), GroEL2 (NR-13790), PstS1 (NR-13790) antibodies from BEI Resources, NIAID, NIH at 1:500 (4 °C, overnight). This is followed by membrane incubation in HRP-labelled secondary antibodies (goat anti-mouse Ig (1:1000), swine anti-rabbit IgG (1:10,000), and rabbit anti-human IgG (1:5000)) (RT, 1 h). Finally, membranes were incubated in Luminata Forte Western HRP Substrate (Millipore; Bedford, MA, USA) (RT, 1 min) before imaging (CL-Xposure Film; Thermo Fisher Scientific). Membranes were washed thrice in PBS-0.05% Tween-20 between each incubation step.
3
Western Blot Analysis of Protein Samples
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Samples were prepared in NuPAGE LDS Sample Buffer (Invitrogen, NP0007) supplemented with NuPAGE Sample Reducing Agent (Invitrogen, NP0004) and heated at 70°C for 10 min. Equal amounts of protein were loaded and resolved on NuPAGE Bis-Tris 4 to 12% gel (Invitrogen, NP032A) and then transferred to nitrocellulose membrane contained in iBlot Gel Transfer Stacks (Invitrogen, NM040319-01) using an iBlot Gel Horizontal Transfer Device (Invitrogen, IB21001). After blotting, membranes were incubated with primary antibodies, diluted in TBST containing 5% skimmed milk, at 4°C overnight. Subsequently, membranes were washed with TBST and incubated with IR700 and IR800 secondary antibodies (LI-COR Biosciences, Lincoln, NE) for 2 hours at room temperature. After further washes, immunoreactive bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
4
Quantitative Protein Immunoblotting Analysis
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50 × 106 cells were lysed for 30 min at 4°C in lysis buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 10 mM NaCl) supplemented with protease and phosphatase inhibitor cocktail tablets (Roche) and samples were centrifuged at 13,000 rpm for 30 min at 4°C. Protein concentrations of supernatants were determined using Bio-Rad protein assay (BIO-RAD). Equivalent protein extract (∼80μg) for each sample was separated by SDS-PAGE and transferred to nitrocellulose membrane using Iblot Gel Transfer stacks and Iblot system (Invitrogen). Membranes were blocked in TTBS (137 mM NaCl, 2 mM KCl, 25 mM Tris and 0.1% tween 20) supplemented with 5% non-fat milk and incubated with primary antibodies against Cleaved Notch1 (Val1744) Antibody (Cell signaling Technology) or Actin (clone I-19, Santa Cruz Biotechnology Inc.) overnight at 4°C with agitation. The fluorescent secondary antibodies, Anti-rabbit or -goat conjugated to CF770 (Biotium) were added for 1 hr at room temperature. Immunoblots were revealed using an Odyssey infrared imaging system (Li-Cor Biosciences) and stripped using Restore Western blot stripping buffer (Pierce).
5
Inflammasome Activation Profiling in aSAH
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Total cell extracts were prepared from monocytes of aSAH patients at day 1 (n = 6) and NC (n = 3) using RIPA buffer supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific). Equal amounts of protein from each sample were separated by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and blotted onto iBlot Gel Transfer Stacks (Invitrogen). These nitrocellulose membranes were then probed with anti-NLRP3 mouse mAb (119–14,885; Ray Biotech), anti-caspase-1 mouse mAb (MAB6215; R&D Systems), and anti-β actin antibody (Abcam, UK) to control for protein loading, followed by a horseradish peroxidase (HRP)-conjugated secondary anti-mouse (Cell Signaling, UK). Antibody binding was detected by enhanced chemiluminescence (ECL) (Amersham-Pharmacia-Biotech, UK).
6
Protein Separation and Immunodetection
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Protein separation by SDS-PAGE was carried out using Mini Protean TGX gels (Bio-Rad) in Tris/glycine/SDS running buffer (Bio-Rad) at 250 V. The gels were stained with Coomassie Brilliant Blue staining solution with Precision Plus Protein (All Blue) as the prestained ladder (Bio-Rad). The iBlot Dry Blotting System (Invitrogen) with nitrocellulose membranes was used for immobilizing proteins on nitrocellulose membranes (iBlot Gel Transfer Stacks, nitrocellulose mini). Enhanced chemiluminescence was performed using the Bio-Rad ChemiDoc XRS+ Imaging System. The blotted membrane was incubated with Clarity Western ECL Substrate, applying an exposure time between 1 and 20 s (depending on sample concentration). Note that non-covalently bound heme is lost during gel electrophoresis. The ECL detection reagent relies on the hydrogen peroxide-mediated oxidation of luminol to the light-emitting 3-aminophthalate in the presence of a catalyst like heme.
7
3D Cell Culture Lysis and Immunoblotting
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Cells were lysed in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X 100, 0.5 sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 20 mM β-glycerophosphate plus Complete protease inhibitors (Roche, Basel, Switzerland). For lysates from 3D cultures, cells were cultured on pure matrigel without collagen. Spheroids were isolated after 4 days by dissolving the matrigel in ice cold Cell Recovery Solution (BD). After centrifugation the spheroids were lysed in RIPA buffer. Lysates were clarified by centrifugation. Equal amounts of protein were separated by SDS–PAGE (NuPAGE® Novex Bis-Tris Gel; Invitrogen) and transferred to nitrocellulose membrane (iBlot®Gel Transfer Stacks; Invitrogen). Alternatively lysates were loaded on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Roth). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.1% Tween-20 and incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. Visualization was done with ECL detection system (Pierce, Rockford, IL, USA). Alternatively detection was done with IR-labled secondary antibodies IRDye 800 CW goat anti-mouse IgG (Licor Biotechnology, Bad-Homburg, Germany) and IRDye 680 LT goat anti-rabbit IgG (Licor Biotechnology).
8
Purification of Malayan Pit Viper Venom
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The venom was a pooled sample obtained from three adult N. sumatrana captured in central Malaysia (Negeri Sembilan) and was supplied by Snake Valley (Seremban, Malaysia).
Resource S ion exchange column and HiTrap Protein A HP affinity column were purchased from GE Healthcare (New Jersey, USA). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate was obtained from Abnova, Taipei, Taiwan. Lichrosphere WP 300 C18 reverse-phase column cartridge was purchased from Merck, New Jersey, USA. iBlot Gel Transfer stacks and iBlot blotting system were supplied by Invitrogen. Sephadex G-25 gel beads and all other reagents were purchased from Sigma – Aldrich (St. Louis, USA) or as stated in the methods.
Resource S ion exchange column and HiTrap Protein A HP affinity column were purchased from GE Healthcare (New Jersey, USA). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate was obtained from Abnova, Taipei, Taiwan. Lichrosphere WP 300 C18 reverse-phase column cartridge was purchased from Merck, New Jersey, USA. iBlot Gel Transfer stacks and iBlot blotting system were supplied by Invitrogen. Sephadex G-25 gel beads and all other reagents were purchased from Sigma – Aldrich (St. Louis, USA) or as stated in the methods.
9
Western Blot Protein Analysis Protocol
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Cells were lysed in RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5 mM PMSF, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 20 mM β-glycerophosphate plus Complete protease inhibitors without EDTA (Roche)] and lysates were clarified by centrifugation (16,000 × g, 10 min). Protein concentration was determined by Bio-Rad DC protein assay. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roth, Karlsruhe, Germany). Alternatively, lysates were loaded on 4–12% NuPAGE® Novex Bis–Tris gels (Invitrogen) and transferred to nitrocellulose membranes (iBlot®Gel Transfer Stacks; Invitrogen). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.05% Tween-20 and incubated with primary antibodies, followed by HRP-labeled secondary antibodies for ECL-based (Pierce, Rockford, IL) visualization with the Amersham600 system (GE Healthcare) or the Fusion Solo (VilberLourmat). Original western blots of all cropped blots are provided as Supplementary Information.
10
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 0.1% [w/v] SDS, 1% NP40, 0.25% [v/v] sodium deoxycholate, 150 mM NaCl, 0.5 mM PMSF, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 20 mM β-glycerophosphate and cOmplete™, EDTA-free Protease Inhibitor Cocktail [Roche]) for 15 min on ice. The lysate was then centrifuged at 16.100 xg, 4 °C, 15 min and the resulting supernatant was quantified using the DC Protein Assay (Bio-Rad). For protein expression analysis, the clear lysate was separated on a 4–12% Bis-Tris Gel (NuPAGE, Thermo Fisher) followed by transfer onto a nitrocellulose membrane using an iBlot® device (iBlot®Gel Transfer Stacks; Invitrogen). The membranes were blocked with 0.5% (v/v) blocking reagent (Roche) in PBS containing 0.05% (v/v) Tween-20 and 0.01% (v/v) Thimerosal and then incubated with primary antibodies, overnight at 4 °C, followed by 1 h incubation with HRP-conjugated secondary antibodies at room temperature. The chemiluminescent signal was detected using an AmershamTM Imager 600 device (GE Healthcare) followed by quantification of the 16-bit images in the linear range using the inbuilt ImageQuant TL 8.1 software.
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