Total protein was extracted from cells or tissues by adding a mixture of RIPA lysis buffer (Solarbio, China) and protease inhibitor (Boster, China) at 4 °C with centrifugation at 12,000 rpm for 15 min. The supernatant was collected, and protein quantification was performed using the BCA kit (Solarbio, China). Proteins were denatured at 95 °C, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to PVDF membranes (Meck, USA). After blocking with 5% skimmed milk powder at room temperature for 2 h, overnight incubation with primary antibodies at 4 °C, and subsequent incubation with secondary antibodies at room temperature for 2 h, membranes were washed with TBST containing 0.1% Tween (Solarbio, China). ECL chemiluminescent solution (Millipore, USA) was applied, and protein expression levels were analysed using the ChemiDocTM imager (Bio-Rad, USA).
Ecl chemiluminescent solution
Manufactured by Merck Group
Sourced in United States
ECL chemiluminescent solution is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It produces a luminescent signal when exposed to the enzyme-labeled target proteins, which can be captured and analyzed using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Boster Bio (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Bca kit, by Solarbio (1 mentions)
Ab32391, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab13420, by Abcam (1 mentions)
Ab39973, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab75745, by Abcam (1 mentions)
Ab56023, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Gel imaging analysis system, by Bio-Rad (1 mentions)
Ab68418, by Abcam (1 mentions)
Ab8049, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
3 protocols using ecl chemiluminescent solution
1
Protein Extraction and Western Blot Analysis
2
Quantifying Osteogenic Markers in MC3T3-E1 Cells
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After the induction of high glucose and treatment of catalpa, proteins in MC3T3-E1 cells were extracted with RIPA lysis buffer (Beyotime, China) and protein concentration was determined by Bradford method. Each hole was loaded with 30μg protein, separated with the SDS-PAGE gel for electrophoresis and transferred to nitrocellulose membranes. The 5% skim milk solution was prepared with the solvent of PBST, which was used to seal the nitrocellulose membranes at room temperature for 2h. Then, primary antibodies including RUNX2 (ab192256;Abcam), Collagen I (ab34710;Abcam), OCN (ab13420;Abcam), BMP4 (ab39973;Abcam), BMP7 (ab56023;Abcam), KDM7A (A14692;ABclonal), β-catenin (ab16051;Abcam), pGSK3β (ab75745;Abcam), GSK3β (ab32391;Abcam), histone (ab1220;Abcam) and GAPDH (ab9485;Abcam) (dilution,1:1000) were incubated with nitrocellulose membranes at 4°C for 12h. PBST was used to wash the nitrocellulose membranes 3 times with 10 min/time. HRP-labeled secondary antibody was incubated with the nitrocellulose membranes at room temperature for 1h. After the nitrocellulose membranes were fully washed, they were then treated with ECL chemiluminescent solution from Millipore. The Bio-Rad gel imaging analysis system was used to collect and analyze the bands.
3
Exosome Protein Profiling by Western Blot
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SDS-PAGE was used to separate the proteins from exosomes and animals. Electrophoretic transfer was performed to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) with TSG 101 (1:1000 dilution; #ab125011, Abcam, MA, USA), CD9 (1:1000 dilution; #ab92726, Abcam, MA, USA), CD63 (1:1000 dilution; #ab68418, Abcam, MA, USA), MAP2 (1:5000 dilution, #17490-1-AP, Proteintech, Chicago, IL, USA), SYN (1:1000 dilution, #ab8049, Abcam, MA, USA), PSD95 (1:1000 dilution; #2507S, CST, MA, USA), NeuN (1:5000 dilution; #ab177487, Abcam, MA, USA), Bax (1:2000 dilution; #BS6420, Bioworld, MN, USA), bcl2 (1:500 dilution; #BS1511, Bioworld, MN, USA), caspase3 (1:500 dilution; #BS7073, Bioworld, MN, USA) overnight at 4 °C. Incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (#ZB2305 or #ZB2301) (1:10,000 dilution; Zhongshan Jinqiao, Beijing, China). Finally, immunoblot assays were performed using ECL chemiluminescent solution (#WBKlS0100, Millipore, MA, USA). Western blot quantitative analysis was performed using Image-Pro Plus 6.0.
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