Tissue samples were lysed with modified RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease and phosphatase inhibitor cocktails (Roche). Protein concentrations were determined using BCA kit (Thermo Scientific). Protein samples were separated by 4-15% SDS-PAGE and transferred to nitrocellulose membranes using Trans-Blot Turbo transfer system (BioRad). Membranes were then blocked with 5% skim milk in TBS and incubated with primary antibodies overnight at 4°C. Membranes were then washed with TBST and incubated with HRP-linked anti-rabbit IgG (7074, Cell Signaling) for 1 h and washed with TBST. Immunoreactivities were assessed using ECL plus kit (Perkin Elmer). Membranes were stripped using stripping buffer (Clontech) and reused for β-actin detection (4970, Cell Signaling).
Ecl plus kit
Manufactured by PerkinElmer
Sourced in United States
The ECL PLUS kit is a chemiluminescent detection system used for the quantitative analysis of proteins in Western blotting applications. The kit provides a sensitive and reliable method for the detection of target proteins labeled with a primary antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated goat anti rabbit antibody, by Jackson ImmunoResearch (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Amersham imager, by Cytiva (3 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Image studio lite software, by LI COR (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Roti block, by Carl Roth (2 mentions)
β actin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
β actin detection, by Cell Signaling Technology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Amersham imager 600, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Tau46, by Cell Signaling Technology (1 mentions)
18 protocols using ecl plus kit
1
Western Blotting of Lysed Tissue Samples
2
Quantitative Protein Analysis of Wnt Signaling
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Five μg of total proteins were resolved by SDS-PAGE under reducing conditions. Immunodetection was performed with antibodies specific to: Active β-catenin, phosphorylated (p) Lrp6, p-Erk, total Erk, Tcf1, Lrp6, and Tubulin [(CST8814, RRID:AB_11127203 ) (CST2568, RRID:AB_2139327 ) (CST9101, RRID:AB_331646 ), (CST9102, RRID:AB_330744 ), (CST2203, RRID:AB_2199302 ), (CST3395, RRID:AB_1950408 ), (CST2125, RRID:AB_2619646 ) Cell Signaling, Beverly, MA, USA] GAPDH and Actin [(SC32233, RRID:AB_627678 ) and (SC47778, RRID:AB_626632 ) Santa Cruz, Santa Cruz, CA, USA]. Immunoreactivities were assessed using ECL plus kit following the manufacture’s protocol (Perkin Elmer, Waltham, MA, USA). Quantification was performed using Image J (RRID:SCR_003070 ) Protein levels were normalized to the levels of housekeeping protein or total protein in within the same sample.
3
Quantifying P2Y12R in Mouse Brain
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Mouse brain homogenates were obtained from rTg4510 mice at 2–6 months of age (male, n = 5) and from non-transgenic mice at 2–6 months of age (male, n = 5) according to the previous report (Shimojo et al., 2020 (link)). Briefly, forebrains including cerebral cortices and hippocampi were homogenized in 20 mM HEPES-NaOH, pH7.4, 150 mM NaCl, 2 mM EDTA supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma) by tissue grinder, and lysed by the addition of SDS-PAGE sample buffer with further resuspension using a 26 G needle. The extracted proteins were separated by SDS-PAGE with pre-cast 10% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Nakarai, Japan) and transferred onto nitrocellulose membranes (BioRad Laboratories, Hercules, CA). After blocking with 5% Skim Milk (in TBS with 0.05% Tween-20), the membranes were incubated with anti-mouse P2Y12R antibody (1:5000) with or without antigen peptide (final concentration 1 µg/mL). Secondary antibody, peroxidase-conjugated goat anti-rabbit antibody (1:5000; 111-035-144, Jackson ImmunoResearch, West Grove, PA), was reacted with membranes and visualized by Amersham Imager 600 (Cytiva, Marlborough, MA) using the enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer). Quantitative analysis was performed with Image StudioTM Lite software (LI-COR, Lincoln, NE).
4
Quantitative Protein Analysis of HepG-2 and BEL-7402 Cells
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HepG-2 and BEL-7402 cells were seeded in six-well plates (2 × 105 per well) and treated with dasatinib and/or SN38 for 48 h. After drug treatment, cells were harvested and resuspended in lysis buffer (50 mmol l−1 Tris-HCl, 150 mmol l−1 NaCl, 2 mmol l−1 EDTA, 2 mmol l−1 EGTA, 25 mmol l−1 NaF, 25 mmol l−1β-Sodium Glycerophosphate, 0.3% NP-40, 0.3% Triton X-100, 0.25% Leupeptin, 0.1% PMSF, 0.1% NaVO3). Tumour tissue samples were sonicated in lysis buffer. All the samples were then centrifuged at 13 200 r.p.m. at 4 °C for 30 min. Protein samples were separated on Tris-glycine gels and transferred to nitrocellulose membrane and then incubated with corresponding HRP-linked antibody. Finally, protein bands were detected with the ECL-Plus Kit (PerkinElmer, MA, USA) and visualised on autoradiography film.
5
Western Blot Analysis of Protein Extracts
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Fractionated tissue extracts were dissolved in SDS-sample buffer containing β-mercaptoethanol (2.5%). Heat-treated samples (55 °C for 15 min) were separated by gel electrophoresis on 10% Tris-glycine SDS-PAGE gels containing a 15-well comb (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes (BioRad Laboratories, Hercules, CA, USA). After blocking with 5% nonfat milk (dissolved in TBS with 0.1% Triton-X100), the membranes were incubated with various antibodies, washed to remove excess antibodies, and then incubated with peroxidase-conjugated goat anti-rabbit antibodies (1:5000; Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse IgG (1:5000; Jackson ImmunoResearch, USA). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer, Waltham, MA, USA). Western blot immunoreactivity was visualized by Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).
6
Western Blot Protein Expression Analysis
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Cells were lyzed in a lysis buffer and BCA protein assay (Pierce) measured homogenized protein concentration. Equal amounts of the total proteins were loaded into 10% SDS-polyacrylamide gel electrophoresis. After being transferred to a nitrocellulose membrane, the membranes were blocked with Roti-Block (Carl Roth, Karlsruhe, Germany). Primary antibodies (Table 1 ) were incubated at 4 °C overnight, followed by a secondary antibody (anti-mouse IgG, 1:5000, anti-rabbit IgG, 1:5000, or anti-goat IgG, 1:5000, Sigma) incubation at room temperature for two hours. The protein bands were developed on film while using a Western detection ECL-plus kit (PerkinElmer, Waltman, MA, USA). ImageJ software was used for band densitometry analysis.
7
Western Blot Analysis of Phospho-JNK
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Tumors were dissected and homogenized in Tris lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40). The samples were run on 10% SDS-PAGE gels (Bio-Rad) and western blots were performed according to standard methods. In brief, lysates were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% milk, the membranes were incubated first with polyclonal antibodies against phosphor-JNK (Calbiochem) or tubulin (DSHB), and then with a secondary HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch). Signals were detected with an ECL-plus kit (PerkinElmer).
8
Western Blot Analysis of Lung Proteins
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Lung slices were lysed in RIPA buffer, and homogenized protein concentrations were measured by BCA protein assay (Pierce, Thermo Scientific, Landsmeer, Netherlands). Equal amounts of total protein were loaded into 8% SDS–polyacrylamide gel electrophoresis. After transferring to a nitrocellulose membrane, primary antibodies were incubated at 4°C overnight, followed by secondary antibody (anti‐mouse, 1:5000 or anti‐rabbit, 1:5000) incubation at room temperature for 1 h. Protein bands were developed on film using Western detection ECL‐plus kit (PerkinElmer, Waltman, MA). ImageJ software was used for band densitometry analysis.
9
Tau Protein Quantification in Alzheimer's
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1 μl sample of S1 and P3 isolated from 24 weeks old rTg4510 and non-transgenic littermate (non-tg) mice or from four pooled Alzheimer’s disease (AD) and healthy control (HC) brains was spotted on nitrocellulose membranes. S1 and P3 isolated from mice was used at a 1:10 dilution. After blocking with a blocking solution containing 5% nonfat milk and 0.1% Triton-X100 in TBS, the membranes were incubated with 1 µg/mL C5.2 overnight at 4 °C. Membranes were washed, and incubated with a peroxidase-conjugated anti-mouse antibody (1:5000; Jackson ImmunoResearch, West Grove, PA). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; Perkin Elmer).
10
Western Blot Analysis of AD and HC Cortices
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One microliter of samples S1 and P3 isolated from AD and HC cortices or from rTg4510 and tTA brains were applied to nitrocellulose membranes. After blocking with 5% nonfat milk and 0.1% Triton X-100 in Tris-buffered saline, the membranes were incubated with 1 μg/mL D1.2 or C10.2. Membranes were washed and incubated with peroxidase-conjugated anti-mouse antibody (1:5000; Jackson ImmunoResearch). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; Perkin Elmer).
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