Cells were lysed, and samples were incubated at 4°C for 30 min in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.5 mM EDTA). Proteins were separated by SDS-PAGE using 4–20% Novex Tris-Glycine gradient Gels (Life Technologies) and electroblotted onto nitrocellulose membranes using the iBLOT2 transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in TBS/0.1% Tween-20 at 4°C overnight. Membranes were then incubated with primary antibodies at room temperature for 1 h. After three washes with TBS-T, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000) at room temperature for 1 h prior to image development using ECL reagents (Abcam, Cambridge, MA, USA).
Ecl reagent
Manufactured by Abcam
Sourced in United States, United Kingdom
ECL reagent is a luminescent substrate used for the detection of proteins in Western blotting applications. It produces a chemiluminescent signal when reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Iblot transfer system, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Nupage bis tris gradient gel, by Thermo Fisher Scientific (3 mentions)
Ab205718, by Abcam (3 mentions)
Ripa buffer, by Solarbio (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab32503, by Abcam (2 mentions)
Sds page, by Beyotime (2 mentions)
Quantity one, by Bio-Rad (2 mentions)
Ab194583, by Abcam (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
30 protocols using ecl reagent
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Cancer Cell Lines
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MCF-7 and MDA-MB 231 cells treated and/or transfected were harvested in PBS and lysed in 1% Triton lysis buffer supplemented in the presence of sodium orthovanadate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride (PMSF), all purchased from Sigma‒Aldrich, USA. The supernatant containing the protein was collected and concentrated by ultracentrifugation, and the protein concentration was measured using the BCA protein assay kit (Bio-Rad, USA). To evaluate the expression of PKM2 (58 kDa), ER(α) (59 kDa), ER(β) (59 kDa), and LDHA (38 kDa), proteins were separated on Tris-Glycine gradient polyacrylamide gels and transferred onto Immuno-Blot PVDF membranes (Bio-Rad). Membranes were incubated in blocking buffer, probed with antibodies specific for PKM2 (Cell Signaling, 1:1000 dilution), β-actin (Cell Signaling, 1:1000 dilution), LDHA (Cell Signaling, 1:1000 dilution), ER(α) (Cell Signaling, 1:1000 dilution), and ER(β) (Sigma, 1:500 dilution) at 4 °C overnight, washed, and then incubated with the appropriate peroxidase-conjugated secondary antibodies (Cell Signaling, 1:2000 dilution) at room temperature. Antibody binding was detected by incubation with enhanced chemiluminescence (ECL) reagents (Abcam) and exposure of the membrane in an ECL machine. The expression of the desired protein was compared to that of β-actin, which served as an internal control.
3
Western Blot Protein Separation and Detection
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Cells were lysed, and samples were incubated at 4°C for 30 min in RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.5 mM EDTA). Proteins were separated by PAGE using 6% to 12% NuPAGE Bis-Tris gradient gels (Life Technologies) and electroblotted onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS at 4°C for 30 min. Membranes were then incubated with primary antibodies (listed in the Supplemental Information ) at room temperature for 3 hr. After three washes with PBS, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000) at room temperature for 1 hr to develop the image using enhanced chemiluminescence (ECL) reagents (Abcam).
4
Western Blot Analysis of Cell Signaling
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Proteins were extracted by RIPA lysis buffer (Beyotime) with 1% phosphatase and protease inhibitors and separated by 10% SDS-PAGE (EpiZyme, Cambridge, MA, USA). Then, we transferred the proteins onto PVDF membranes (Pierce Biotechnology, Waltham, MA, USA) and blocked the membranes with 5% milk at 37 °C for 1 h. The membranes were incubated with primary antibody overnight at 4 °C. A subsequent 2 h incubation in secondary antibody was performed, and the membranes were then exposed to ECL reagents (Abcam, Cambridge, UK). Antibodies and dilutions were as follows: RAB20 antibody (Abcam, ab197209, 1:1000); α-tubulin (CST, #2144, 1:1000); β-actin (CST, #3700, 1:1000); CDK2 (CST, #2546, 1:1000); CyclinE1 (CST, #20808, 1:1000); CyclinD1 (CST, #55506, 1:1000); CyclinB1 (CST, #12231, 1:1000); cdc2 (CST, #9116, 1:1000); Chk1 (CST, #2360, 1:1000); cdc25C (CST, #4688, 1:1000); Phospho-cdc25C (CST, #4901, 1:1000); p53 (CST, #2527, 1:1000); Phospho-p53 (CST, #9286, 1:1000); and p21Waf1/Cip1 (CST, #2947, 1:1000).
5
Western Blot Analysis of Inflammatory Signaling
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Cells were lysed in RIPA lysis buffer (Beyotime) containing 1% phosphatase and protease inhibitors at 4 °C for approximately 30 min. Protein (30 μg) was separated by 10% SDS-PAGE (PAGE-Gel Fast Preparation Kit, EpiZyme) and transferred onto PVDF membranes (Pierce Biotechnology). Protein bands were blocked with 5% nonfat milk at room temperature for 2 h and incubated with the indicated primary antibody overnight at 4 °C and the appropriate secondary antibody for 2 h. ECL reagents (Abcam) were used for exposure. The following antibodies and dilutions were used: AIM2 (ab93015, Abcam, 1:1000), BCL2A1 (CY5582, Abways, 1:1000), p44/42 MAPK (137F5, CST, 1:1000), c-Myc (E5Q6W, CST, 1:1000), α-tubulin (AF0001, Beyotime, 1:1000), NF-κB Pathway Sampler Kit (#9936, CST, 1:1000), Phospho-Erk1/2 Pathway Sampler Kit (#9911, CST, 1:1000), IL-1β (#12703, CST, 1:1000), cleaved IL-1β (#83186, CST, 1:1000) and Caspase-1 (#3866, CST, 1:1000).
6
Western Blot Protein Detection Protocol
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Cells were lysed, and samples were incubated at 4°C for 30 min in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.5 mM EDTA). Proteins were separated by PAGE using 6–12% NuPAGE Bis-Tris gradient Gels (Life Technologies) and electroblotted onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS at 4°C for 30 min. Membranes were then incubated with primary antibodies (Supplemental Data) at room temperature for 1 hr. After three washes with PBS, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 hr to develop the image using ECL reagents (Abcam, Cambridge, MA, USA).
7
Western Blot Protein Detection Protocol
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Cells were lysed, and samples were incubated at 4°C for 30 min in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate and 0.5 mM EDTA). Proteins were separated by PAGE using 6% to 12% NuPAGE Bis-Tris gradient Gels (Life Technologies) and electroblotted onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS at 4°C for 30 min. Membranes were then incubated with primary antibodies (Supplementary Data ) at room temperature for 1 h. After three washes with PBS, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 h to develop the image using ECL reagents (Abcam, Cambridge, MA, USA).
8
Protein Expression Analysis Protocol
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The protein assay, sample preparation, and SDS-PAGE electrophoresis were performed as described in our previous investigations [41 (link),85 (link),86 (link),87 (link)]. Briefly, the cells were harvested and lysed with NP-40 lysis buffer. Based on the type of protein, 10–30 µg of total protein was subjected to electrophoresis on SDS-PAGE gels (15% for MW 60 KD and lower, and 10% for MW > 60 KD), and separated proteins were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad; #1620112). After overnight blocking (5% fat-free milk), the membranes were incubated with primary antibodies (P-H2A (Ser 139), GRP78, IRE-1, ATF-6, XBP-1s, e-IF2α, p-eIF2α (Ser 51), caspase-3, Beclin-1, LC3β-II, p62, and GAPDH) overnight at 4 °C. Antibodies were used in dilutions according to the manufacturer’s protocol. After incubation with suitable secondary antibodies for 90 min at room temperature, the membranes were incubated with enhanced chemiluminescence (ECL) reagents (Abcam, Cambridge, MA, USA) and developed by the ChemiDocTM MP imaging system (Bio-Rad, Hercules, CA, USA). The intensity of blots was measured by Image Lab densitometry software, and all bands were normalized to the GAPDH protein amount to correct for marginal deviations in protein loading.
9
Western Blot Protein Analysis
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Cells were lysed, and samples were incubated at 4°C for 30 min in RIPA buffer. Proteins were separated on 4–12% NuPAGE Bis-Tris gradient gels containing SDS (ThermoFisher Scientific) and electroblotted onto PVDF membranes using the iBLOT transfer system (ThermoFisher Scientific). The membranes were blocked with 5% non-fat dry milk in PBS at 4°C for 30 min. Membranes were then incubated with primary antibodies at room temperature for 1 h or at 4°C overnight. After three washes with PBS, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 h, followed by image development using ECL reagents (Abcam) and quantification of protein levels using ImageLab (Bio-Rad).
10
Western Blot Analysis of Protein Expression
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Total proteins were obtained from the transfected SRA01/04 cells and analyzed by western blot. Briefly, the proteins were separated through SDS-PAGE and immediately transferred onto polyvinylidene difluoride membranes (Millipore, USA), followed by the blockage of membranes by 5% nonfat milk for 1 h. Next, the membranes were incubated with primary antibodies against E2F3, Bax, Bcl-2, cleaved caspase 3, E-cadherin, N-cadherin, vimentin, and α-SMA (Abcam, USA) and HRP-conjugated secondary antibody (Sangon). Eventually, the immunized signals were determined by the enhanced chemiluminescence (ECL) reagent (Abcam) and ImageLab software version 4.1 (Bio-Rad Laboratories, USA) was applied for image acquisition and densitometry analysis of the blots in this assay according the description of a previous study (28 (link)). The intensity of bands was measured as the total volume under the three-dimensional peak, which could be observed in two dimensions using the "Lane Profile" tool to correct the width of the band, accounting for the area under the shaded peak of interest.
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