HCT116 cells were harvested, lysed on ice in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) and boiled. Protein concentration was measured by BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The protein samples were separated by SDS-PAGE and transferred to the membrane (Pall, New York, NY, USA). Afterwards, the membrane was immunoblotted with the corresponding primary antibody (C-parp (#5625), C-cas9 (#9505), C-cas3 (#9661), Bcl-2 (#4223), Bax (#5023), SHP2 (#3752), P-Erk1/2 (#9101) and Erk1/2 (#9102), 1:1000, Cell Signaling Technology, Danvers, MA, USA; Tubulin (#ET1602-4), 1:5000, HUABIO, Hangzhou, China) and the appropriate HRP-conjugated secondary antibody, and determined with ECL detection reagent (Thermo Scientific, Waltham, MA, USA).
Ecl detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Austria
ECL detection reagents are chemiluminescent substrates used in Western blotting to detect and visualize target proteins. They produce a luminescent signal when combined with an enzyme-labeled secondary antibody, allowing researchers to quantify and analyze protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (24 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (22 mentions)
Ripa buffer, by Beyotime (11 mentions)
Ripa buffer, by Thermo Fisher Scientific (11 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Pvdf membrane, by Thermo Fisher Scientific (7 mentions)
Hatf00010, by Merck Group (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Bca kit, by Beyotime (6 mentions)
Stripping buffer, by Thermo Fisher Scientific (5 mentions)
Roti load1 buffer, by Carl Roth (5 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Protease inhibitor cocktail, by Beyotime (4 mentions)
Phosphatase inhibitor cocktail, by Merck Group (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Ab8227, by Abcam (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (4 mentions)
205 protocols using ecl detection reagent
1
Immunoblot Analysis of Cell Signaling
2
SDS-PAGE and Western Blot Analysis
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The plant crude extract or purified plant-produced proteins were further analyzed by SDS-PAGE and Western blot. For crude plant extracts, proteins were separated by SDS-PAGE gradient gel (4–15 %) under non-reducing and reducing conditions using wild type N. benthamiana extract as the negative control. The separated proteins were stained with InstantBlue® (Abcam, Cambridge, UK) for proteins visualization and also transferred to nitrocellulose membrane (Bio-Rad, California, USA) for immunoblotting. The membranes were blocked with 5 % skim milk and probed with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Southern Biotech, Alabama, USA) that was diluted 1:4000 in 3 % skim milk. The membranes were washed three times with 1X PBST (1X PBS with 0.05 % Tween 20 (Sigma-Aldrich, Darmstadt, Germany)) and once with 1X PBS. Then, the membrane was detected for the chemiluminescence signal using ECL detection reagent (Thermoscientific, USA) as per manufacturer's instructions.
3
Western Blot Analysis of Cell Proteins
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Total proteins were extracted from cells and separated using 10% SDS-PAGE (80 V for 1 h and 120 V for the subsequent 2 h) and then electrophoretically transferred (300 mA for 1.5 h) to a nitrocellulose membrane (Millipore; HATF00010). The membrane was blocked with 5% milk in PBST for 1 h and incubated with primary antibodies (1:1000) at 4 °C overnight and secondary antibodies (1:2000) for 1 h at room temperature. Target proteins were detected with enhanced chemiluminescence (ECL) detection reagent (Thermo Scientific; 34075). The antibodies used in the study were anti-DNMT3a antibody (Abcam; ab13888), anti-HDAC1 antibody (Abcam; ab7028), anti-ACAT1 antibody (Abcam; ab168342), anti-FAK antibody (BD; 610088), anti-β-actin antibody (CST; 12262), anti-AMPKα antibody (CST; 2532), and anti-phospho-AMPKα antibody (CST; 2535).
4
SDS-PAGE and Western Blotting
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Proteins were extracted from cell lysate. 10% SDS-PAGE was applied to separate the proteins according to their molecule weight. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). Then the membrane was blocked with 5% milk, and followed by incubation with respective primary antibody and secondary antibody. ECL detection reagent (Thermo Scientific, Rockford, IL, USA) was employed to detect the proteins.
5
Immunoblotting for Protein Analysis
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The MV4-11 cells or NB4 cells were first treated, harvested and lysed. Proteins from cell lysate were separated with 10% SDS-PAGE gel and then transferred to a nitrocellulose membrane (HATF00010, Millipore, Billerica, MA, USA). The membrane transfer was conducted in a cold room, on ice for 90 min with a constant current of 300 mA. The membrane was then blocked with 5% non-fat milk in PBST for 1 h at room temperature with gentle shaking. Subsequently, the membrane was washed 3 times with PBST and incubated overnight with the primary antibody. Following successive washing with PBST, the membrane was incubated with the corresponding secondary antibody for 1 h at room temperature. After washing, proteins were detected with an ECL detection reagent (34075, Thermo Scientific, Rockford, IL, USA).
6
Western Blot Analysis of Collagens
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Cell lysates were loaded onto acrylamide gels and transferred to nitrocellulose membrane. Membranes were blocked and incubated with the following antibodies according to manufacturer's protocols: polyclonal rabbit anti-mouse Collagen I α 1, rabbit anti-mouse Collagen III α 1 (both Novus Biologicals) and polyclonal rabbit anti-mouse ß-Actin, rabbit anti-mouse Smad3, and rabbit monoclonal anti-mouse Phospho-Smad3 (Ser 423/425, clone C25A9) (Cell Signaling Technology).
Membranes were then exposed to anti-rabbit horseradish peroxidase as secondary antibody (Dako), and signal detection was performed using ECL detection reagent (Thermo Fisher).
Membranes were then exposed to anti-rabbit horseradish peroxidase as secondary antibody (Dako), and signal detection was performed using ECL detection reagent (Thermo Fisher).
7
Protein Extraction and Detection Protocol
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Proteins were extracted from different cells by using cell lysis buffer with protease and phosphatase inhibitors (Solarbio, Beijing, China) according to the manufacturer’s instructions, and protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane (Millipore, USA). Membranes were incubated with antibodies, and the ECL detection reagent (Thermo Fisher Scientific) was used to detect signal. Detailed antibodies information was listed in Supplementary Table 1 .
8
Western Blotting Analysis of hUC-MSCs
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Western blotting analysis was conducted as we described before [6 (link), 25 (link)]. Briefly, the hUC-MSCs at various passages (P3, P6, P15) were lysed with Laemmli sample buffer (BioRad) and inactivated in 100 °C for 5 min. Then, the samples were electrophoresed in SDS-PAGE gel and transferred onto a PVDF membrane (Life Sciences). After blocking in 5% nonfat milk (BD) for 1 h, the membrane was incubated with primary antibody (Cell Signaling, Abcam) and HRP-conjugated secondary antibody (GE Healthcare). Finally, the membrane was incubated with ECL Detection Reagent (ThermoFisher) and transferred into Super-signal West Pico Chemiluminescent Substrate (Prierce) for development. The antibodies were listed in Additional file 7 : Table S3.
9
Protein Expression Analysis by Western Blot
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Protein was extracted using RIPA lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) and the protein concentration was measured with a protein BCA assay kit (Nanjing KeyGen Biotech Co., Ltd.). Subsequently, 30 µg protein was separated on a 12% SDS-PAGE gel by electrophoresis and transferred to 0.45 µm PVDF membranes (Merck KGaA). The membranes were immersed in a blocking solution containing 5% non-fat dry milk at room temperature for 2 h and then incubated overnight at 4°C with an antiserum containing antibodies against LC3 (1:1,000; cat. no. 2775; Cell Signaling Technology, Inc.), p62 (1:1,000 dilution; cat. no. 5114; Cell Signaling Technology, Inc.) ATG5 (1:500 dilution; cat. no. 10181-2-AP; ProteinTech Group, Inc.) and ATG12 (1:500 dilution; cat. no. 10088-2-AP; ProteinTech Group, Inc.). Subsequently, the membranes were washed and incubated with a peroxidase-conjugated secondary antibody (1:3,000 dilution; cat. no. TA130023; OriGene Technologies, Inc.) at room temperature for 2 h. Finally, the protein bands were visualized with an ECL detection reagent (Thermo Fisher Scientific, Inc.) and the results were quantified with the use of ImageJ software J2x (Rawak Software Inc.). An anti-GAPDH antibody (1:1,000 dilution; cat. no. 2118; Cell Signaling Technology, Inc.) was used as a control and the results were presented as the ratio of density of target protein to the GAPDH value.
10
Protein Immunoblotting Procedure
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