Western blotting was carried out according to the previous report with modifications (Zhao et al., 2019 (link); Shi et al., 2014 (link)). The expression levels of PI3K, p‐Akt, p‐JAK2, p‐STAT3, Bax, Bcl‐2, Caspase‐3, and β‐actin after the treatments were measured. Briefly, LoVo cells (1 × 106 cells/well) were seeded in a six‐well plate, followed by incubation overnight at 37°C with 5% CO2. Then the cells were treated for 24 h with LRPS, LPAC, and LRPS&AC, respectively. After that, cytoplasmic and nuclear protein were extracted using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, USA) according to the operation manual. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) was used to separate the proteins, which were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The loaded membranes were blocked with 5% skimmed milk in Tris‐buffered saline with Tween‐20 (TBST) buffer for 1 h at room temperature, followed by incubation with the primary antibodies at 4°C on a shaker overnight. The membranes were then washed twice and incubated at room temperature for 1 h with the corresponding secondary antibody conjugated with horseradish peroxidase. The blots were detected by enhanced chemiluminescence (ECL) detection kits (Bio‐Rad, Hercules, USA) and analyzed using Empiria Studio® software.
Ecl detection kit
Manufactured by Bio-Rad
Sourced in United States, Germany
The ECL detection kit is a laboratory equipment designed for the detection and visualization of proteins or other biomolecules in Western blot analysis. The kit contains the necessary reagents and solutions to facilitate the chemiluminescent detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Image lab software, by Bio-Rad (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
β actin, by Merck Group (5 mentions)
Quantity one software, by Bio-Rad (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Anti p stat3, by Cell Signaling Technology (3 mentions)
Laemmli buffer, by Bio-Rad (3 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Specific horseradish peroxidase conjugated goat anti rabbit or goat anti mouse secondary antibody, by Bio-Rad (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Gel doc xr system, by Bio-Rad (2 mentions)
81 protocols using ecl detection kit
1
Western Blotting Analysis of Signaling Pathways
2
Western Blot Analysis of Brain Samples
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The selected brain samples (frontal cortex, hippocampus, and hypothalamus) were collected after the behavior test and EEG recording. Samples were lysed with RIPA buffer (ELPIS Biotech Inc., Daejeon, Republic of Korea). The proteins were separated by 12% SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk, and were incubated at 4 °C with the primary antibodies (1:1000) overnight and with the secondary antibodies (1:2000) for 1 h. The proteins were detected using enhanced chemiluminescence (ECL) detection kits (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of BV2 Cells
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BV2 cells were collected and homogenized using RIPA lysis buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1.0 mM EDTA at a pH of 7.4) supplemented with 50X Protease Inhibitor Cocktail; Roche Diagnostics) on ice. The protein concentration was determined using the BCA assay (Applygen Technologies, Inc.). The total protein (20 µg/per lane) from each sample was separated using 8% SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes. After 1 h of blocking with 5% non-fat milk at room temperature, the membranes were incubated with primary antibodies against COX-2 (1:1,000; cat. no. A3560; ABclonal Biotech Co., Ltd.), HIF-1α (1:1,000; cat. no. 36169; Cell Signaling Technology, Inc.) and β-actin (1:5,000; cat. no. A2228; Sigma-Aldrich; Merck KGaA) overnight at 4˚C. After washing three times, the membranes were incubated with anti-mouse/rabbit IgG HRP-conjugated secondary antibodies (1:2,000; cat. no. 7076 and 7074, respectively; Cell Signaling Technology, Inc.) for 2 h at room temperature. Protein bands were visualized using an ECL detection kit (Bio-Rad Laboratories, Inc.). Quantification of the band intensities was performed using ImageJ Software (version 1.8.0; National Institutes of Health) and normalized to the band intensities of β-actin.
4
Protein Extraction and Western Blot Analysis
5
Western Blot Analysis of Protein Expression
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Cells were grown on 6-well culture plates and were treated with BBSKE, oxaliplatin, or the combination for the indicated times. The cells were then washed twice or three times with 1 mL of PBS, and were lysed using cell lysis buffer. For western blot analysis, equal amounts of protein in each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane. The membranes were blocked using 5 % nonfat milk at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. Then, the membranes were washed three times with TBST for 10 min each time, and incubated with the peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were visualized using an ECL detection kit (Bio-Rad Laboratories, CA, USA) and the images were captured and analyzed with the ChemiDoc Imaging Systems.
6
Western Blot Analysis of Bax and Bcl-2
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Western blot was done as described previously [25 (link)]. Briefly, approximately 20 μg (20 μl) protein per gel well was loaded and resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The SDS-PAGE gel was transferred to polyvinylidene difluoride membranes. The membranes were incubated in tris-buffered saline (T-TBS) containing 3% non-fat dry milk and a specific proportion of the primary antibodies for bax and bcl-2(Cell signaling Technology) overnight at 4°C. The blot was then washed and incubated with goat anti-mouse IgG conjugated to peroxidase (Origene, America). Antibody binding was detected by chemoluminescence staining using the ECL detection kit (Bio-Rad). The density of each band was quantified by densitometry of Bandscan 5.0 software.
7
Western Blot Analysis of Molecular Targets
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Western blotting was performed as described previously [41 (link)]. The specific first antibodies GAPDH (1 : 1000), p-AKT (1 : 1000), p-CREB (1 : 1000), CREB (1 : 1000), AKT (1 : 1000), COX-2 (1 : 1000), TNF-α (1 : 1000), HO-1 (1 : 1000), Nrf2 (1 : 1000), iNOS (1 : 1000), and Histone H3 (1 : 5000) were purchased at CST (Cell Signaling Technology, INC). After three times of wash with TBST (pH = 7.2), for 10 minutes each, membranes were incubated with horseradish peroxidase-conjugated secondary mouse anti-rabbit IgG (1 : 15,000 dilutions) for 1 hour at room temperature. The membranes were washed three times, 10 minutes/time; the immunoreactive bands were then detected using ECL Detection Kit (Bio-Rad Laboratories, Richmond, California, USA), and labelling was visualized by Image Lab software (Bio-Rad).
8
Western Blot Analysis of Hamster Brain
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Western blot analysis was performed for the same day slaughtered animals (100th DPI) and terminally dead hamsters. We prepared 10% brain homogenate for biochemical tests. The homogenization of frozen brain samples was carried out in RIPA buffer containing a cocktail of protease inhibitors and then sonicated for 15 s, centrifuged at 20,000× g for 5 min. The obtained supernatants were collected and then boiled for 10 min after the addition of loading buffer (250 mMTris-HCl pH 6.8, 10% SDS, 0.5% BPB, 50% glycerol, 0.5 M DTT). The protein concentration of each brain sample was calculated before adding loading sample buffer by means of BCA assay (CWBio, Beijing, China) and approximately 30 µg/wel of the protein extract was subjected to western blotting. Samples were separated via SDS-PAGE and the proteins were transferred to PVDF membranes (Immobilon-PSQ, ISEQ00010, 0.2 μm). Blots were blocked by 5% non-fat milk in TBST (25 mMTris base, 137 mM sodium chloride, 2.7 mM potassium chloride and 0.05% Tween-20, pH 7.4) for 1 h at room temperature, incubated with the indicated primary antibody overnight at 4 °C, and the corresponding HRP-labeled secondary antibody for 50 min at 37 °C, and the signal detected using an enhanced chemiluminescence (ECL) detection kit (Bio-Rad, Hercules, CA, USA).
9
Comprehensive Western Blotting Protocol
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Western blotting was carried out as described previously (X. Xu et al., 2020 (link)). Primary antibodies used in this study including rabbit anti‐YAP (ab205270, Abcam, 1: 1, 000), mouse anti‐YAP (WH0010413M1, Sigma, 1:1, 000), rabbit anti‐p‐YAP (#13008, CST, 1:1, 000), rabbit anti‐LATS1 (#3477, CST, 1:1, 000), rabbit anti‐p‐LATS1 (ser909) (#9157, CST, 1:1, 000), rabbit anti‐MST1 (#3682, CST, 1:1, 000), rabbit anti‐p‐MST1/2 (Thr183/Thr180) (#49332, CST, 1:1, 000), rabbit anti‐SAV1 (#13301, CST, 1:1, 000), rabbit anti‐p‐MOB1 (Thr35) (#8699, CST, 1:1, 000), rabbit anti‐p53 (bs‐2090R, Bioss, 1:1, 000), rabbit anti‐Lamin B1 (ab16048, Abcam, 1: 1, 000), mouse anti‐CDK6 (#3136T, CST, 1:1, 000), rabbit anti‐FOXM1 (AV39518, Sigma, 1: 1, 000), and mouse anti‐PHD1 (F5303, Sigma, 1: 1, 000). Mouse anti‐β‐actin (A5316, Sigma, 1:10, 000) or rabbit anti‐GAPDH (#2118, CST, 1:5, 000) was used as a loading control. The protein signals were detected using ECL detection kit (Bio‐Rad) and analyzed using Quantity One software (Bio‐Rad).
10
Stromal Cell Protein Expression Profiling
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Protein extract was performed on stromal cell cultures and tissue samples obtained from fertile women in the proliferative and secretory phase, and subsequently lysed and isolated. Protein concentrations were then measured using a BCA Protein Assay Kit (Boster, China). Equal amounts of denatured protein were separated via electrophoresis in 10% SDS polyacrylamide gels, then transferred to polyvinyl difluoride membranes (Millipore, Billerica, USA). These membranes were saturated with blocking buffer for 1 h. Following this, membranes were incubated with rabbit polyclonal antiLHCGR (1:1,000 dilution; Proteintech), rabbit polyclonal anti-HOXA10 (1:1,000 dilution; Proteintech), rabbit polyclonal anti-ITGB3 antibodies (1:2,000 dilution; Proteintech), rabbit polyclonal anti-LIF antibodies (1:1,000 dilution; Proteintech) and mouse monoclonal anti-MECA-79 antibodies (1:500 dilution; Santa Cruz) at 4 °C. The blots were then incubated with HRP-conjugated anti-rabbit IgG for 2 h. Finally, proteins were detected by the enhanced chemiluminescence (ECL) detection kit (Bio-Rad, USA) and visualized using the film exposure. Then analyzing gels and western blots with ImageJ.
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