Total protein was extracted from tissues/cells by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The concentration of extracted protein was determined using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Same amount (40 μg) of protein samples were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred onto a nitrocellulose membrane (EMD Millipore). Then, the membranes were blocked using tris-buffered saline containing 5% skimmed milk at room temperature for 2 hours, followed by overnight incubation using corresponding primary antibodies: MTSS1 (1:500; cat. no. sc-101204; Santa Cruz Biotechnology Inc) or GAPDH (1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology Inc) at 4 °C. Next day, the membranes were further incubated in horseradish peroxidase–conjugated secondary antibody (1:5,000; cat. no. sc-2371; Santa Cruz Biotechnology Inc) at room temperature for 2 hours. The signals were visualized with an enhanced chemiluminescence protein detection kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc), and then the blots were quantified using Image J (NIH).
Enhanced chemiluminescence protein detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced chemiluminescence protein detection kit is a laboratory equipment product designed for the detection and visualization of proteins in Western blot analysis. It utilizes chemiluminescent technology to provide a sensitive and quantitative method for protein detection.
Automatically generated - may contain errors
3 protocols using enhanced chemiluminescence protein detection kit
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Total protein was extracted from tissues or cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Protein concentration was evaluated by bicinchoninic acid assay (Beyotime Institute of Biotechnology). Equal amounts (30 µg) of protein samples were separated using 10% SDS-PAGE gel and then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked in tris-buffered saline (TBS) with 5% skimmed milk for 2 h at room temperature, and then were incubated with primary antibodies: MTSS1 (1:500; cat. no. sc-101204) or GAPDH (1:1,000; cat. no. sc-47724; both from Santa Cruz Biotechnology Inc.) at 4°C overnight. The membranes were then incubated with corresponding horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. sc-2371; Santa Cruz Biotechnology Inc.) for 1 h at 37°C, followed by visualization using an enhanced chemiluminescence protein detection kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc). Protein bands were quantified by densitometric analysis using ImageJ software (NIH).
3
Protein Expression Analysis in Glioma Cells
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Total protein was extracted from the glioma cells by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). The concentration of the extracted protein was determined using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Same amounts of protein samples were separated using SDS-PAGE. Thereafter, the proteins were transferred onto nitrocellulose membranes, which were blocked using tris-buffered saline tween with 5% skimmed milk at room temperature for 1 h. This was followed by incubation using corresponding primary antibodies (anti-β-actin (dilution 1:1000), anti-Flag (dilution 1:5000), anti-ERK5(CST, cat#3552, dilution 1:1000), anti-PRPS1 (dilution 1:1000), anti-PRPS2 (dilution 1:1000), anti-PPAT (dilution 1:1000), ant-TKTL1 (dilution 1:1000)) at 4 °C overnight. Next day, the membranes were incubated with anti-mouse or anti-rabbit IgG (dilution 1:10,000) antibodies at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence protein detection kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc), and the signal was quantified by Image J software (NIH, Bethesda, MD, USA).
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