RIPA buffer mixed with protease inhibitor (Thermo Fisher Scientific, USA) was used to lyse cells for harvest of total protein. The protein was separated by 4–20% Precast-Gel (Solarbio, China) and transferred to a PVDF membrane (Millipore, USA). Then 5% bovine serum albumin was used to block the membrane for 1 h at room temperature, and incubated with primary antibody overnight at 4℃. The following primary antibodies were used: anti-Flag (Origene, USA), anti-SATB2 (Abcam, USA), anti-β-catenin, anti-active β-catenin (Cell Signaling Technology, USA), anti-β-actin (Zhongshanjinqiao, China), anti-RUNX2 (Biorbyt, England), anti-JHDM1D (Abcam, USA), anti-DKK1 (Santa Cruz, USA), anti-H3K9me2, anti-H3K27me2 (Abcam, USA). HRP-conjugated secondary antibody (1:10,000; Zhongshanjinqiao, China) was then used to incubate the membrane for 1 h at room temperature. At last, we used ECL and Super Signal detection reagents (Thermo Fisher Scientific, USA) to detect the membrane.
Supersignal detection reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal detection reagents are a series of chemiluminescent substrates designed for the detection of proteins in Western blotting applications. The reagents produce a luminescent signal when interacting with the target protein, allowing for sensitive and quantitative detection using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Proteojet mammalian cell lysis reagent, by Thermo Fisher Scientific (2 mentions)
Rabbit monoclonal anti p53, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti flag, by OriGene (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti satb2, by Abcam (1 mentions)
Precast gel, by Solarbio (1 mentions)
Anti dkk1, by Santa Cruz Biotechnology (1 mentions)
Anti active β catenin, by Cell Signaling Technology (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit monoclonal anti ago2, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti gapdh, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit and goat anti mouse secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Mouse polyclonal anti gapdh, by Cell Signaling Technology (1 mentions)
Rabbit polyclonal anti phospho p53, by Cell Signaling Technology (1 mentions)
4 protocols using supersignal detection reagents
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of S1P Receptors
3
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were obtained using the ProteoJET Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in TBST (50 mM Tris (pH 7.5), 200 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membranes were then incubated with the primary antibody in the blocking solution for 1 h at room temperature, washed three times with TBST for 15 min, incubated with the HRP-conjugated secondary antibody at room temperature for 1 h and then washed three times with TBST. Antigen-antibody complexes were detected using the SuperSignal detection reagents (Thermo Scientific, Rockford, IL, USA). The following antibodies were used: rabbit monoclonal anti-p53, rabbit monoclonal anti-AGO2, rabbit monoclonal anti-GADD45A, mouse monoclonal anti-GAPDH (Cell Signalling Technology, MA, USA) and horseradish peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse secondary antibodies (Santa Cruz. CA, USA).
4
Western Blot Analysis of p53 Signaling
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Whole-cell lysates were obtained using the ProteoJET™ Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat dried milk in TBST (50 mM Tris [pH 7.5], 200 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membrane was then incubated with primary antibody in the same concentration of milk in TBST for 1 h at room temperature, washed three times with TBST for 15 min, and then incubated with the HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was again washed three times with TBST. Antigen-antibody complexes were detected using the SuperSignal detection reagents (Thermo Scientific, Rockford, IL, USA). The following antibodies were used: rabbit monoclonal anti-p53, rabbit polyclonal anti-Phospho-p53, mouse polyclonal anti-GAPDH, mouse polyclonal anti-ACTIN (Cell Signaling Inc. Danvers, MA, USA); secondary antibodies (goat-anti-rabbit and goat-anti-mouse) conjugated to horseradish peroxidase (Santa Cruz. CA, USA).
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