For western blotting, the telencephalon of 6 month-old fish were dissected or 9 day-old larvae were sacrificed according the local ethical regulations. The telencephalon or whole larvae were used for protein isolation using 200 µL RIPA buffer (Sigma, R0278) with addition of phosphatase (Roche, Catalog Number 04906837001) and protease inhibitors (Roche, Catalog number 04963132001). 5 µl of protein ladder (ThermoFisher, #26634) and 10 µl of total protein was loaded in 4–12% Bis-Tris precast gradient gels (NuPage, Catalog number NP0322BOX) and the gels were run in NuPAGE MES SDS running buffer (Novex, Life technologies, NP0006-1) at 200 V for 60 min. Blots were transferred to methanol activated PVDF (Novex, Life technologies, LC2002) membrane, and were blocked in 10% milk powder in 0.2% Tween in 1X-PBS for 1h at room temperature. The primary antibodies at appropriate dilutions in 2 ml 0.2% Tween in 1X-PBS were applied overnight at 4 °C in 15 or 50 ml plastic containers. Following the washing steps, secondary antibodies at 1:4000 dilutions (anti-rabbit IgG HRP (Santa Cruz, #sc-2004) or anti-mouse IgG peroxidase (Sigma, #A8924)) were applied at room temperature for 2 h. Gel images were acquired by ImageQuant LAS4000 (GE Healthcare) using Western BLoT Ultra Sensitive HRP Substrate (Takara, #T7104A).
Western blot ultra sensitive hrp substrate
Manufactured by Takara Bio
Sourced in United Kingdom
Western BLoT Ultra Sensitive HRP Substrate is a chemiluminescent detection reagent that can be used to detect and quantify proteins on Western blots. The substrate reacts with horseradish peroxidase (HRP)-conjugated secondary antibodies to produce a luminescent signal, allowing for highly sensitive protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Cell Signaling Technology (3 mentions)
Western blot quant hrp substrate, by Takara Bio (2 mentions)
Anti caspase 1 3866, by Cell Signaling Technology (2 mentions)
Hrp goat anti mouse superclonal igg, by Thermo Fisher Scientific (2 mentions)
Anti il 1β h153, by Santa Cruz Biotechnology (2 mentions)
Anti asc al 177, by Adipogen (2 mentions)
Anti nlrp3 clone cryo 2, by Adipogen (2 mentions)
Anti β actin clone ac 15, by Merck Group (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protein ladder, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg peroxidase, by Merck Group (1 mentions)
Anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ibright analysis software, by Thermo Fisher Scientific (1 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin hrp antibody, by Merck Group (1 mentions)
Wb stripping solution strong, by Nacalai Tesque (1 mentions)
11 protocols using western blot ultra sensitive hrp substrate
1
Western Blotting of Telencephalon Proteins
2
Quantitative Analysis of HA-TRIM5 Expression
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The pelleted cells were lysed in 2 × Bolt LDS sample buffer (Thermo Fisher Scientific, Cat# B0008) containing 2% β-mercaptoethanol (Bio-Rad, Cat# 1610710) and incubated at 70 °C for 10 min. Expression level of HA-tagged TRIM5 in CRFK cells was confirmed by Western blotting using an anti-HA Tag (6E2) mouse mAb (HRP Conjugate) (CST, Cat# 2999S, × 5,000). After stripping of the membrane using WB Stripping Solution Strong (Nacalai Tesque, Cat# 05677–65), the membrane was re-probed with an anti-β-Actin–HRP antibody (Sigma-Aldrich, Cat# A3854-200UL, × 20,000) as a loading control. Chemiluminescence was detected using Western BLoT Ultra Sensitive HRP Substrate (TaKaRa, Cat# T7104A) according to the manufacturer’s instructions. Bands were visualized using an iBright FL1500 imaging system (Thermo Fisher Scientific), and the band intensity was quantified using iBright analysis software (Thermo Fisher Scientific).
3
Immunoblot Analysis of Inflammasome Proteins
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Samples were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min, the membranes were incubated overnight at 4°C with the following primary antibodies (Abs): anti-ASC (AL-177; Adipogen), anti-β actin (clone AC-15; Sigma), anti-caspase-1 (3866; Cell Signaling Technology), anti-IL-1β (H153; Santa Cruz Biotechnology), anti-NEK7 (EPR4900; abcam, Cambridge, UK), and anti-NLRP3 (clone Cryo-2; Adipogen). As secondary Abs, HRP-goat anti-mouse Superclonal IgG (Thermo Fisher Scientific) or HRP-goat anti-rabbit IgG (Cell Signaling Technology) was incubated with membrane for 1 hr. After being washed with TBS-Tween, immunoreactive bands were visualized using Western BLoT Quant HRP Substrate (Takara Bio) or Western BLoT Ultra Sensitive HRP Substrate (Takara Bio).
4
Western Blot Analysis of Mitochondrial Proteins
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Western blot analyses as well as the removal of bound antibodies were conducted as described previously [9] (link), [49] (link). Proteins were separated by SDS-PAGE, blotted onto a PVDF membrane and reacted with antibodies against Grx1 (1:800), Grx2 (rabbit, 1:200 or guinea pig, 1:500), cTXNPx (1:3000), Tpx (1:2000), LipDH (1:1000), ASCT (1:500), and mtTXNPx (1:1000). The secondary goat antibodies were diluted 1:10,000 (guinea pig) and 1:20,000 (rabbit). Bands were visualized by chemiluminescence using the Super-Signal West Pico Kit (Pierce) or the Western BLoT Ultra Sensitive HRP Substrate (Takara).
5
Western Blot Analysis of Protein Markers
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Western blot analysis was performed as described previously17 (link),47 (link),48 (link). Briefly, cell lysates were prepared using RIPA buffer (20 mM Tris, 2.5 mM EDTA, 1% Triton X, 10% glycerol, 1% deoxycholic acid, 0.1% SDS, 50 mM NaF, and 10 mM Na4P2O7·10H2O), and subjected to SDS–PAGE. The proteins were electrophoretically transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS-T for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies, followed by incubation for 1 h with secondary antibodies conjugated to horseradish peroxidase (HRP). Immunoreactive bands were visualized using a Western BLoT Ultra Sensitive HRP Substrate (Takara Bio Inc.). The expression level of β-actin served as an internal control for protein loading. Primary antibodies against ASC45 (link), VE-cadherin (Abcam, Cambridge. MA), and β-actin (Sigma-Aldrich) were used. HRP-goat anti-rabbit IgG (Cell Signaling Technology Inc., Boston, MA) and HRP-goat anti-mouse IgG (Cell Signaling) were used as secondary antibodies.
6
Immunoprecipitation of Sycp2 in Testes
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Testes from 8–14 fish were homogenized in 5 ml of RIPA buffer. After freezing at -80°C and sonication, the soluble fraction was recovered by centrifugation at 20,000 x g for 15 min at 4°C. The concentrations of the protein extracts were determined with a Protein Assay Lowry Kit (Nacalai). Immunoprecipitation was performed from ~2.5 mg of testis proteins of each genotype with either 25 μg of guinea pig anti-Sycp2 antibody or 25 μg of normal guinea pig IgG. The antigen-antibody complex was recovered with 50 μμl of protein A Sepharose beads (GE Healthcare). The immunoprecipitated samples were eluted in 40 μl of 2x sample buffer at 95°C for 10 min and migrated on a 7.5% Mini-PROTEAN TGX precast protein gel (Bio-Rad) at 200 V for 30 min. After transfer to an Immobilon-P membrane (Millipore) at 100 V for 70 min, immunoblotting was performed with a rat anti-Sycp2 antibody and a goat anti-rat HRP-conjugated antibody (Santacruz) using Can Get Signal Immunoreaction Enhancer Solution (Toyobo). The signals were developed with Western BLoT Ultra Sensitive HRP Substrate (Takara) and captured with an ImageQuant LAS 4000 Mini (GE Healthcare).
7
Protein Expression Analysis of Cell Aggregates
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Cell aggregates treated with or without inhibitors from day 5 were dissociated using TrypLE Express Enzyme (Thermo Fisher Scientific) on day 6 and lysed in radioimmunoprecipitation assay buffer containing 50-mM Tris hydrochloride (pH 7.6), 150-mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease inhibitor cocktail (Nacalai) and ethylenediaminetetraacetic acid-free phosphatase inhibitor cocktail (Nacalai). A total of 2.7–9.0 μg of protein extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membrane (Merck, Immobilon). Three biological replicates were analyzed. The following primary antibodies were used at the indicated dilutions: anti-beta-actin (rabbit, 1:1,000; Cell Signaling, 4970), anti-beta-catenin (rabbit, 1:1,000; Cell Signaling, 8480), anti-phospho-Smad1/5 (rabbit, 1:1,000; Cell Signaling, 9516), and anti-phospho-Smad2 (rabbit, 1:1,000; Cell Signaling, 3108). Anti-rabbit IgG, HRP-linked antibody (rabbit, 1:2,000; Cell Signaling, 7074) was used as a secondary antibody. The blocking buffer used was Western BLoT Immuno Booster (TaKaRa). The chemiluminescent substrate used was Western BLoT Ultra-Sensitive HRP Substrate (TaKaRa). The chemiluminescent signal was detected using LAS 4000mini (Cytiva) and analyzed using ImageJ.
8
Histone Extraction and Western Blot Analysis
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ESCs were lysed with RIPA lysis buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% IGEPAL CA-630 (NP-40 substitute; MP Biomedicals, Solon, OH), 150 mM NaCl, and 50 mM Tris-HCl (pH 8.0)). Histone proteins were isolated by acid extraction protocol. Protein fractions were treated at 99 °C for 5 min in SDS sample buffer containing 80 mM DTT, subjected to SDS-PAGE, and blotted onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA). The primary and secondary antibodies used in this study were listed in Supplementary Table 2 . The blots were developed using the ECL Prime Western Blotting Detection Kit (GE Healthcare, Chicago, IL) or the Western BLoT Ultra Sensitive HRP Substrate (Takara, Otsu, Japan) and visualized using X-ray films or the ChemiDoc XRS system (BioRad, Hercules, CA). Signal intensity of each band on the blots was measured and quantified with Image Lab Software (BioRad).
9
Gedatolisib Inhibition Assay by WB
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Cell lysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were performed as previously described with minor modifications (31 (link),32 (link)). The protein concentrations of each lysate were quantified using a Protein Quantification kit (Takara Bio, Inc.). The primary antibodies and secondary antibody used in the present study are listed in Table SI . The proteins were visualized using the Western BLoT Ultra-Sensitive HRP Substrate (Takara Bio, Inc.) and detected using an ImageQuant LAS-4000 mini system (GE Healthcare; Cytiva).
To prepare the cell lysate, 6×104 cells were seeded in 6 cm dishes and cultured for 48 h at 37°C and 5% CO2. For evaluation of the dose-dependent suppression of gedatolisib, the cells were treated with 0.1% DMSO or various concentrations (0.1 nM-10 µM) of gedatolisib for 24 h. For evaluation of the time-dependent suppression of gedatolisib, the cells were treated with 0.1% DMSO or gedatolisib (100 nM or 10 µM) for 0, 4, 8, 24 and 48 h. For immunoblotting analysis of p-NDRG1 (Thr346), the cells were treated with 0.1% DMSO or 100 nM gedatolisib for 4 h.
To prepare the cell lysate, 6×104 cells were seeded in 6 cm dishes and cultured for 48 h at 37°C and 5% CO2. For evaluation of the dose-dependent suppression of gedatolisib, the cells were treated with 0.1% DMSO or various concentrations (0.1 nM-10 µM) of gedatolisib for 24 h. For evaluation of the time-dependent suppression of gedatolisib, the cells were treated with 0.1% DMSO or gedatolisib (100 nM or 10 µM) for 0, 4, 8, 24 and 48 h. For immunoblotting analysis of p-NDRG1 (Thr346), the cells were treated with 0.1% DMSO or 100 nM gedatolisib for 4 h.
10
SDS-PAGE and Western Blot Analysis
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Samples were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min, the membranes were incubated overnight at 4ºC with the following primary antibodies (Abs): anti-ASC (AL-177; Adipogen), anti-β actin (clone AC-15;
Sigma), anti-Caspase-1 (3866; Cell Signaling Technology), anti-IL-1β (H153; Santa Cruz Biotechnology), and anti-NLRP3 (clone Cryo-2; Adipogen). As secondary Abs, HRP-goat antimouse Superclonal IgG (Thermo Fisher Scientific) or HRP-goat anti-rabbit IgG (Cell Signaling Technology) was incubated with membrane for 1 h. After being washed with TBS-Tween, immunoreactive bands were visualized using Western BLoT Quant HRP Substrate (Takara Bio)
or Western BLoT Ultra Sensitive HRP Substrate (Takara Bio).
Sigma), anti-Caspase-1 (3866; Cell Signaling Technology), anti-IL-1β (H153; Santa Cruz Biotechnology), and anti-NLRP3 (clone Cryo-2; Adipogen). As secondary Abs, HRP-goat antimouse Superclonal IgG (Thermo Fisher Scientific) or HRP-goat anti-rabbit IgG (Cell Signaling Technology) was incubated with membrane for 1 h. After being washed with TBS-Tween, immunoreactive bands were visualized using Western BLoT Quant HRP Substrate (Takara Bio)
or Western BLoT Ultra Sensitive HRP Substrate (Takara Bio).
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