Cells were lysed by RIPA buffer containing protease inhibitor cocktail (MedChem Express). Protein concentrations were determined by BCA Protein Assay Kit (KeyGENBioTECH). Equal amount of protein was loaded and separated by SDS–PAGE, and transferred onto PVDF membranes (Bio-Rad Laboratories). Membranes were incubated with primary antibodies against FOXM1 (1:100, Santa Cruz), β-actin (1:2000, Cell Signaling Technology) and H3K27me3 (1:1000, Abcam) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The ChemiDoc MP system was employed to detect the protein signals via chemiluminescent substrate ECL kit (Merck Millipore).
Chemiluminescent substrate ecl kit
Manufactured by Merck Group
Sourced in China
The Chemiluminescent substrate ECL kit is a laboratory equipment product designed to detect and quantify proteins in western blot analysis. The kit utilizes a chemiluminescent substrate to generate a light signal proportional to the amount of target protein present in the sample. This allows for sensitive and specific detection of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Keygen Biotech (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
H3k27me3, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
2 protocols using chemiluminescent substrate ecl kit
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis of SJYLF-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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SJYLF-treated HCT-8 cells (0.5–1.5
mg/mL extract) were lysed in an ice-cold radioimmunoprecipitation
assay (RIPA) lysis buffer (Beyotime, Beijing, China) having phosphatase
inhibitors (Merck Millipore, MA) and protease inhibitor cocktails
(Merck Millipore, MA) for about 30 min. Lysed cells were centrifuged
(6000g, 15 min) at 4 °C, and the clear supernatant
was obtained.
Bicinchoninic acid (BCA) kit was used to check
protein concentration. Proteins were denatured by adding a 5×
loading buffer to 20 μL of sample followed by sample boiling
for 5 min at 90 °C. Tris–glycine SDS-PAGE (12%, 120 V)
was used to separate the protein. Proteins were then transferred to
poly(vinylidene fluoride) (PVDF) membrane (Millipore, MA) by electroblotting.
To avoid nonspecific binding, 5% bovine serum albumin (BSA) in TBS/T
buffer was used as a blocking buffer. After washing with PBS, membranes
were incubated overnight with primary antibodies of β-actin,
Bcl-2, Bax, CDK4, and cyclin D1 at 4 °C. After primary incubation,
horseradish peroxidase-conjugated secondary antibody (Zsbio, Beijing,
China) was added, and membranes were again incubated for 1 h. Chemiluminescent
substrate ECL kit (Merck Millipore, MA) was used to detect the protein
bands that were visualized by using X-ray film exposure.
mg/mL extract) were lysed in an ice-cold radioimmunoprecipitation
assay (RIPA) lysis buffer (Beyotime, Beijing, China) having phosphatase
inhibitors (Merck Millipore, MA) and protease inhibitor cocktails
(Merck Millipore, MA) for about 30 min. Lysed cells were centrifuged
(6000g, 15 min) at 4 °C, and the clear supernatant
was obtained.
Bicinchoninic acid (BCA) kit was used to check
protein concentration. Proteins were denatured by adding a 5×
loading buffer to 20 μL of sample followed by sample boiling
for 5 min at 90 °C. Tris–glycine SDS-PAGE (12%, 120 V)
was used to separate the protein. Proteins were then transferred to
poly(vinylidene fluoride) (PVDF) membrane (Millipore, MA) by electroblotting.
To avoid nonspecific binding, 5% bovine serum albumin (BSA) in TBS/T
buffer was used as a blocking buffer. After washing with PBS, membranes
were incubated overnight with primary antibodies of β-actin,
Bcl-2, Bax, CDK4, and cyclin D1 at 4 °C. After primary incubation,
horseradish peroxidase-conjugated secondary antibody (Zsbio, Beijing,
China) was added, and membranes were again incubated for 1 h. Chemiluminescent
substrate ECL kit (Merck Millipore, MA) was used to detect the protein
bands that were visualized by using X-ray film exposure.
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