SGC-7901 cells (1.5 × 105 cells/well) in the logarithmic growth phase were cultured in 6-well cell culture plates for 24 h and then treated with different concentrations of CBMP for 24 h. We used RIPA mixed with protease and phosphatase inhibitors to lyse to gain total proteins of SGC-7901 cells. The BCA protein assay kit (Fudebio, Hangzhou, China) was used to measure protein concentration. The extracted protein was packed and stored at −80°C. We used 10% or 12% SDS-PAGE to separate proteins and then transfer them onto polyvinylidene difluoride (PVDF) membranes (Millipore, Atlanta, USA). 5% skim milk was used to block PVDF membranes at room temperature for 1 h, and then, primary antibodies were incubated overnight in a refrigerator at 4°C. After that, the PVDF membranes were incubated with secondary antibodies for 2 h at room temperature. The signals were detected by ImmobilonWestern Chemiluminescent HRP Substrate (Millipore, Atlanta, USA), and the images of the band were evaluated using ImageJ software.
Bca protein assay kit
Manufactured by Fudebio
Sourced in China
The BCA protein assay kit is a laboratory tool used to quantify the total protein concentration in a sample. It relies on the bicinchoninic acid (BCA) method, which involves the reduction of Cu2+ to Cu+ by proteins in an alkaline medium. The resulting purple-colored reaction product is then measured spectrophotometrically, allowing for the determination of the total protein content in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Protease inhibitors and phosphatase inhibitor, by Roche (1 mentions)
Radioimmunoprecipitation assay (ripa), by Roche (1 mentions)
Pvdf membrane, by BD (1 mentions)
Skim milk, by BD (1 mentions)
Fdr007, by Fudebio (1 mentions)
5 protocols using bca protein assay kit
1
Western Blot Analysis of CBMP-Treated SGC-7901 Cells
2
Protein Extraction from A375 Cells
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A density of 1 × 105 A375 cells were seeded onto 6-well plates. After 24 h, cells were treated with varying concentrations of CP for 12 h, 24 h and 48 h. Then cells were washed twice immediately with pre-chilled PBS on ice. The cytoplasmic proteins were lysed using RIPA mixed with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was measured by the BCA protein assay kit (Fudebio, Hangzhou, China). The protocol we used was that same with our previous publication [38 (link)].
3
Protein Expression Analysis of Panc-1 Cells
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Panc-1 cells were seeded into 6-well plates at a density of 1 × 106 cells/well with varying concentrations of CP. After 48 h treatment, cells were washed twice with pre-cold PBS twice on ice. Proteins were lysed with RIPA (Roche, Basel, Switzerland). Collecting and vortexed the cell lysate at 12,000 rpm for 10 min, then put on ice for 10 min to remove the cell debris. Mixed the cellular protein with sample loading buffer (Fudebio, Hangzhou, China), then boiled the mixture at 95 °C for 10 min. The BCA protein assay kit (Fudebio, Hangzhou, China) was used to measure the protein concentration, and 12% SDS-PAGE (Fudebio, Hangzhou, China) was used to separate the protein, which was then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Blocked PVDF membranes with 5% skim milk (BD, NY, USA) dissolved in TBST solution for 1 h at room temperature, which were then incubated the immunoblots with specific primary antibodies overnight at 4 °C. After primary antibody binding, incubating the blots with AP-conjugated anti-rabbit secondary antibodies for 1 h at room temperature. Beta-tublin was used as the internal control, and the immunoblots were developed with the ECL method.
4
Western Blot Analysis of Mouse Lung Tissue
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Mouse lung tissue samples were lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The BCA Protein Assay Kit (Fudebio-tech) was used for protein concentration quantification. Proteins were electrophoresed on 10% or 12% SDS-polyacrylamide gels and transferred to PVDF membranes (EMD Millipore). Membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with specific primary antibodies. The membranes were washed three times with Tris-Buffered Saline Tween-20 (TBST) and then incubated with secondary antibodies conjugated with HRP (Abcam) for 1 h at room temperature. At last, the signal was scanned using the Tanon-5200 infrared scanning system (anon Science & Technology Co., Ltd., Shanghai, China) after three washes again and quantified using ImageJ software.
5
Western Blot Analysis of Stem Cell Markers
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Whole-cell extracts were prepared using protein lysis buffer (Invent Biotechnologies, Plymouth, MN, USA). A bicinchoninic acid (BCA) protein assay kit (Fudebio, Hangzhou, China) was used to determine the protein concentrations. Total proteins (30 μg) were separated using 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then probed with primary antibodies against CMTM2 (ABP53869, Abbkine, Wuhan, China, 1 : 1000), SRY-box transcription factor 2 (SOX2) (11064-1-AP, Proteintech, Rosemont, IL, USA, 1 : 600), NANOG (67255-1-Ig, Proteintech, 1 : 10000), CD44 (15675-1-AP, Proteintech, 1 : 5000), and GAPDH (60004-1-Ig, Proteintech, 1 : 1000) overnight at 4°C. Subsequently, the membranes were incubated with goat anti-rabbit antibodies diluted with 0.3‰ in TBST (FDR007, Fudebio, Guangzhou, China, 1 : 10000) or goat anti-mouse antibody diluted with 0.3‰ TBST for 1 h at room temperature. The membranes were then scanned using the Tanon 5200 automated image analysis system (Tanon Science and Technology Co., Ltd, Shanghai, China), and the ImageJ software (National Institutes of Health) was used to evaluate the band intensity.
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