Total proteins were harvested through RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) which containing Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA). The quantitation of protein was examined by BCA protein quantification kit. Equal amounts of proteins were subjected to SDS/PAGE electrophoresis, and the separated proteins were transferred onto a PVDF membrane. Blocking the membranes with 5% non-fat milk in 1 x TBST (10 mM Tris-HCl, 150 mM NaCl, pH 8.0, and 0.1% Tween 20) for 30 min at room temperature, then incubated in primary antibody overnight at 4°C. The primary antibodies included: Bcl-2 (1: 2000, Epitmics, USA), Bax (1: 1000, CST, USA), GRP78 (1: 1000, CST, USA), p-PERK (1: 1000, CST, USA), p-eIF2α (1: 1000, CST, USA), CHOP (1: 1000, CST, USA), calpain-1 (1: 1000, CST, USA), calpain-2 (1: 1000, CST, USA) and caspase-12 (1: 1000, CST, USA). Next, the PVDF membrane was washed with TBST solution for 10 min and repeated 3 times. Then, the membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature. The membranes were washed and developed by the ECL chemiluminescent kit (Amersham Biosciences, Piscataway, NJ, USA).
Ecl chemiluminescent kit
Manufactured by Cytiva
Sourced in United States
The ECL Chemiluminescent kit is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. The kit contains reagents that generate a chemiluminescent signal when in contact with the target protein, which can then be detected and measured using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Fst antibody, by Santa Cruz Biotechnology (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Bradford reagent, by Merck Group (2 mentions)
Bca reagent kit, by Applygen (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ab108353, by Abcam (1 mentions)
Ab155095, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab68781, by Abcam (1 mentions)
Anti human gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
8 protocols using ecl chemiluminescent kit
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Cell Signaling
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Western blot was performed as previously described [24 (link)] . The protein lysates of uterine tissues or cells were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocked in nonfat milk, membranes were incubated 16 h at 4 °C with each primary antibody for anti-Aurora A (ab108353, Abcam), anti-PLK1 (phospho T210) (ab155095, Abcam), anti-Tubulin (2144, CST), anti-β-actin (4970, CST), anti-P-STAT3 (9145, CST) or anti-Phospho-cdc2 (Tyr15) (4539, CST), respectively. Membranes were incubated in matched second antibody for 1 h. The ECL chemiluminescent kit (Amersham Biosciences) was used to visualize signals.
3
Quantitative Analysis of OPN and GAPDH
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Western blots were run as previously reported [28] (link). Samples were incubated with primary antibodies for OPN (Biorbyt, California, USA) or GAPDH (Cell Signaling Technology, Boston, USA) and then with matched second antibodies conjugated with horseradish peroxidase. The signals were developed with a ECL chemiluminescent kit (Amersham Biosciences, Boston, USA). All experiments were repeated three times.
4
Quantitative Protein Analysis by Western Blot
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Western blot analysis was performed by Bio-X Vision Biological Technology Co., Ltd. For Western blot analysis, total proteins were isolated from the samples by homogenization in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, and complete protease inhibitor cocktail (Roche). The concentration of proteins was measured by Bradford reagent (Sigma), separated on 10% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore). After blocking in 5% low-fat milk in PBST (0.1% Tween 20 in PBS) for 1 h, the membranes were incubated with GFP antibody (1∶500, Santa Cruz Biotechnology), Fst antibody (1∶500, Santa Cruz Biotechnology) or mouse b-actin antibody (1∶2000, Santa Cruz Biotechnology) overnight at 4°C. After washing in PBST, the membranes were incubated in goat anti-rabbit antibody conjugated with horsera dish peroxidase (1∶5000) for 1 h, followed by three washes in PBST. The signals were detected by ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights).
5
Quantifying Protein Expression in Uterine Tissues
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Proteins were extracted from uterine tissues with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO3, 1% sodium deoxycholate, 1% Triton X-100 and 0.1% SDS). A complete protease inhibitor (Roche Applied Science, Upper Bavaria, Germany) was added into each sample to prevent protein degradation. Protein concentration was measured with BCA reagent kit (Applygen, Beijing, China). Samples were electrophoretically separated on 10% PAGE and electrically transferred onto PVDF membranes. After blocking with 5% non-fat milk containing 0.5% Tween 20 for 1 h, the membrane was incubated with rabbit anti-total STAT3 antibody (1:1000; #9132, Cell Signaling Technology) or rabbit anti-phosphorylated (Tyr 705) STAT3 antibody (1:1000; #9131, Cell Signaling Technology) overnight at 4°C. The membrane was then incubated with HRP-conjugated secondary antibody (1:5000) for 1 h. Signals were analyzed by ECL Chemiluminescent kit (Amersham Pharmacia Biotech, IL USA). Tubulin was used as a loading control.
6
Quantifying Lung Protein Expressions
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Western blot analysis for protein expressions were performed as reported previously in our laboratory [7 (link)]. Lung specimens were collected after mice sacrifice and lysed in 20 mM Tris for following standard western blot analysis. Primary antibodies were list as follow: anti-SP-B antibody (Abcam, Cambridge, MA), anti-SP-C antibody (Abcam, Cambridge, MA). Primary α-tubulin antibody from Beyotime Biotechnology (Shanghai, China) was loaded as the internal control for normalization between different groups. The corresponding secondary antibodies were then incubated and imaged with an ECL+Chemiluminescent Kit (Amersham Bioscience, Arlington Heights, IL) to identify the immune bands. Further analysis was performed by densitometry quantified with Quantity One software (Bio-Rad, Berkeley, CA).
7
Western Blot Analysis of Protein Expression
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For Western blot analysis, total proteins were isolated from the samples by homogenization in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, and complete protease inhibitor cocktail (Roche). The concentration of proteins was measured by Bradford reagent (Sigma), separated on 10% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore). After blocking in 5% low-fat milk in PBST (0.1% Tween 20 in PBS) for 1 h, the membranes were incubated with tGFP antibody (1:500, SantaCruz Biotechnology), Fst antibody (1:500, Santa Cruz Biotech- nology) or mouse GAPDH antibody (1:2000, Santa Cruz Biotechnology) overnight at 4uC. After washing in PBST, the membranes were incubated in goat anti-rabbit antibody conjugated with horseradish peroxidase (1:5000) for 1 h, followed by three washes in PBST. The signals were detected by ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights).
8
Endometrial Tissue Protein Extraction and Analysis
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Whole endometrial tissue lysate was prepared and homogenized in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.25% sodium deoxycholate). Protein concentration was measured and adjusted by using the BCA Reagent kit (Applygen, Beijing, China). Samples were run on a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat dry milk in TBST (0.1% Tween 20 in TBS) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human telomerase (ab68781, Abcam, Cambridge, UK), ER alpha (sc-7207, Santa Cruz, CA, USA) or anti-human Gapdh (sc-25778, Santa Cruz). After three washes in TBST, membranes were incubated with secondary antibodies conjugated with horseradish perioxidase for 1 h at about 25°C. The signals were developed with the ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Films were scanned using a flat-bed scanner. The intensities of the bands representing ER alpha and TERT were evaluated using the Image J analysis software (Rawak Software, Inc., Germany).
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