Total protein of MC3T3 cells were harvested with RIPA lysis buffer and quantified with a BCA assay kit (Beyotime). Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bull serum albumin in Tris buffered saline with Tween 20 and incubated with the following primary antibodies at 4 °C overnight: cyclin D1 (1:500, A2708, ABclonal, Wuhan, China), SP7 (1:1000, ab94744, Abcam, Shanghai, China), OCN (1:650, A5786, ABclonal), Runx2 (1:500, A2851, ABclonal), P27Kip1 (1:500, A2692, ABclonal), and β-actin (1:100000, AC026, ABclonal). Horseradish peroxidase-conjugated anti-rabbit IgG (H + L) secondary antibodies were added (1:3000, AS014, ABclonal) and incubated at 25 °C for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) by Tanon 5200 (Tanon, Shanghai, China).
Ecl chemiluminescence kit
Manufactured by 7Sea Biotech
Sourced in China
The ECL chemiluminescence kit is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to generate a light signal proportional to the amount of target protein present in the sample. The kit provides the necessary reagents to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
A5786, by ABclonal (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ac026, by ABclonal (2 mentions)
A2708, by ABclonal (2 mentions)
A2851, by ABclonal (2 mentions)
A2692, by ABclonal (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Ab94744, by Abcam (2 mentions)
As014, by ABclonal (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Ab23981, by Abcam (1 mentions)
Active β catenin, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
β actin, by ABclonal (1 mentions)
Ac004, by ABclonal (1 mentions)
5 protocols using ecl chemiluminescence kit
1
Western Blot Analysis of Osteoblast Markers
2
Western Blot Analysis of Osteoblast Markers
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Total protein of MC3T3 cells were harvested with RIPA lysis buffer and quantified with a BCA assay kit (Beyotime). Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bull serum albumin in Tris buffered saline with Tween 20 and incubated with the following primary antibodies at 4 °C overnight: cyclin D1 (1:500, A2708, ABclonal, Wuhan, China), SP7 (1:1000, ab94744, Abcam, Shanghai, China), OCN (1:650, A5786, ABclonal), Runx2 (1:500, A2851, ABclonal), P27Kip1 (1:500, A2692, ABclonal), and β-actin (1:100000, AC026, ABclonal). Horseradish peroxidase-conjugated anti-rabbit IgG (H + L) secondary antibodies were added (1:3000, AS014, ABclonal) and incubated at 25 °C for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) by Tanon 5200 (Tanon, Shanghai, China).
3
Osteoclast Differentiation Pathway Regulation
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After treatment with ODN for 7 days under mouse RANKL, the proteins from RAW264.7 cells were harvested using RIPA lysis buffer and quantified with bicinchoninic acid protein assay kit (Beyotime Biotech Inc., Jiangsu, China). The proteins were separated on 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% BSA in TBST and incubated with the following primary antibodies: cyclin D1 (A2708), cyclin E1 (A0112), cyclin A2 (A2891), cyclin B1 (A2056), Nfatc (A15339), c-fos (A0236), RANK (A13382), MMP9 (A2095), OPG (A2100), RANKL (A2550), and β-actin (AC026). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were diluted at 1 : 3000 and incubated at room temperature for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) with Tanon 5200 (Tianneng, Shanghai, China).
4
Analyzing Osteogenic Markers in rBMSCs
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The proteins of rBMSCs were harvested with RIPA lysis buffer and quantified with a bicinchoninic acid protein assay kit. Proteins were separated on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% BSA in TBST and incubated with the following primary antibodies: Runx2 (1:1000, ab23981, Abcam, Hong Kong), ColI (1:650, A5786, ABclonal, Boston, USA), GSK-3β (1:650, A3174), active-β-catenin (1:1000, #8480, Cell Signaling Technology, Danvers, USA), and β-actin (1:8000, AC004, ABclonal, Boston, USA). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were diluted at 1:3000 and incubated at room temperature for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) with a ChemiDoc XRS+ system (Biorad, America).
5
Osteoclastogenesis Signaling Pathway Analysis
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RAW264.7 cells were treated with ODN and RANKL for 4 days and the cell proteins were harvested with RIPA lysis buffer and quantified with a bicinchoninic acid protein assay kit (Beyotime Biotech Inc., Jiangsu, China). Proteins were separated by 12% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bovine serum albumin in Tris-buffered saline-Tween and incubated with the following primary antibodies: Nfatc (A15339), c-fos (A0236), RANK (A13382), MMP9 (A2095), and β-actin (AC026), respectively. HRP-conjugated anti-rabbit secondary antibodies were diluted at 1:3000 and incubated at room temperature for 1h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech) with a Tanon 5200 device (Tianneng, Shanghai, China).
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