To determine the effects of ABE on the expression levels of apoptosis related proteins in PC cells, a Human Apoptosis Array (Raybiotech, GA, USA) was used. The PC cells were cultured in 6-well plates (5 × 105 cells/well) and treated with 2 µM ABE for 24 h. After treatment, the proteins were extracted from the collected pellet and apoptosis array protocol was performed according to the manufacturer instructions. Finally, protein detection was conducted using a chemiluminescence detection system (Syngene, USA).
Chemiluminescence detection system
Manufactured by Syngene
Sourced in United States
The Chemiluminescence detection system is a laboratory instrument used to measure the emission of light during a chemical reaction. It is designed to quantify the presence and amount of specific molecules or analytes in a sample by detecting the light output produced by a chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
1 mm pmsf buffer, by Beyotime (2 mentions)
Human apoptosis array, by RayBiotech (1 mentions)
Cw2383, by CWBIO (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
P0100 1, by Solarbio (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
4 protocols using chemiluminescence detection system
1
Apoptosis Protein Expression in PC Cells
2
Western Blot Analysis of Wear Particle-Stimulated BMDMs
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Following stimulation by wear particles for 1 h and other interventions, BMDMs were lysed using a RIPA buffer (9806S, Cell Signaling Technology) containing PMSF (P0100-1, Solarbio) and a phosphatase inhibitor cocktail (CW2383, CW Biotech). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were cut into small pieces according to different protein molecular masses and then probed with the relevant antibodies overnight at 4°C. Details of the antibodies are listed in Supplementary Table S3 . A chemiluminescence detection system (Syngene, Cambridge, United Kingdom) was used to visualize the protein bands and further analysis was performed using Gene Tools (Syngene, Cambridge, United Kingdom).
3
Protein Quantification and Western Blot
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Total protein was isolated from cultured cells with RIPA lysis buffer (Beyotime, China) and 1 mM PMSF buffer (Beyotime). Then, the protein concentration was determined using a BCA protein assay kit (Beyotime). Samples containing equal amounts of protein were loaded into SDS-PAGE gels. After electrophoresis, proteins in the gels were transferred onto PVDF membranes (Millipore, USA). After blocking with 5% bovine serum albumin at room temperature for 2 h, membranes were incubated with the primary antibodies listed above overnight at 4°C. After several TBST washes and incubation with HRP-conjugated secondary antibodies, bound proteins were detected with ECL reagents (Bio-Rad, USA) in a chemiluminescence detection system (Syngene, USA). The bands intensity was quantified with ImageJ software and normalized to that of GAPDH. The relative band intensity in the control group was set to 1.
4
Protein Isolation and Western Blot Analysis
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Total protein was isolated from cultured cells with RIPA lysis buffer (Beyotime, China) and 1 mM PMSF buffer (Beyotime). Then, the protein concentration was determined using a BCA protein assay kit (Beyotime). Samples containing equal amounts of protein were loaded into SDS-PAGE gels. After electrophoresis, proteins in the gels were transferred onto PVDF membranes (Millipore, USA). After blocking with 5% bovine serum albumin at room temperature for 2 h, membranes were incubated with the primary antibodies listed above overnight at 4°C. After several TBST washes and incubation with HRP-conjugated secondary antibodies, bound proteins were detected with ECL regents (Bio-Rad, USA) in a chemiluminescence detection system (Syngene, USA). The bands intensity was quantified with ImageJ software and normalized to that of GAPDH. The relative band intensity in the control group was set to 1.
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