HL-7702/SRE-Luc cells were plated in 96-well plates at a density of 2.5 × 105 cells per well and cultured for 18 hours. The cells were then switched to medium containing 5% LPDS, 10 μM compactin, and 10 μM mevalonate for 18 hours in the absence or presence of PMFE, and luciferase activity was measured as previously described (10 (link)). Normalized luciferase values were determined by dividing luciferase activity by the protein content in cell extracts quantified using the BCA (bicinchonininc acid) protein assay (Beyotime). Western blot was measured as previously described (35 (link)).
Protein assay
Manufactured by Beyotime
Sourced in China
The Protein Assay is a laboratory equipment used to quantify the total protein concentration in a sample. It employs a colorimetric method to determine the protein content by measuring the absorbance of the sample at a specific wavelength. The Protein Assay provides a reliable and standardized way to measure protein levels in various biological samples, enabling researchers and scientists to analyze and understand protein-related processes and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid (bca), by Beyotime (3 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Nitrocellulose membrane, by Abcam (1 mentions)
Fibronectin, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Bio-Rad (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
E cadherin, by Abcam (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Gadph, by Cell Signaling Technology (1 mentions)
Polyacrylamide gel, by Wuhan Boster Biological Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescent kit, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
5 protocols using protein assay
1
Measuring Luciferase Activity in HL-7702/SRE-Luc Cells
2
Quantitative Protein Analysis of Cell Lysates
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Proteins extracted from cultured cells or tissues were quantitated by a protein assay (Beyotime, Shanghai, China). Cell or tissue lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane (Millipore, Billerica, USA). Following blocking in PBS containing 5 % fat-free milk, the nitrocellulose membrane was incubated with primary antibodies against WT1, E-cadherin, Fibronectin, and Vimentin (Abcam). Blots were stripped and reprobed with β-actin antibody (Santa Cruz Biotechnology, California, USA) as an internal control. After overnight at 4 °C and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h at room temperature, the antigen–antibody immunoreactivity was detected in a sensitive digital imaging equipment (Bio-Rad, Richmond, USA) using a commercial ECL detection kit (Millipore). All experiments were repeated three times with the similar results.
3
BCA Protein Assay and Western Blot
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According to instructions of the BCA (bicinchoninic acid) protein assay (Beyotime Biotechnology Co., Shanghai, China), cell proteins were extracted to determine the protein concentration. Loading buffer was added to the extracted proteins and boiled at 95°C for 10 min. Thirty micrograms of protein sample were loaded into each well of a 10% polyacrylamide gel (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) and separated by electrophoresis. Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with 5% bovine serum albumin (BSA) at room temperature for 1 h. Next, the PVDF membrane was incubated with primary antibodies against, p-PI3K, PI3K, phosphorylated (p)-AKT, AKT, p-mTOR, mTOR, LC3, Beclin1, p62 and GADPH (Cell Signaling Technology, Inc.) at 1:1000 dilutions overnight at 4°C. After three washes with Tris-buffered saline containing Tween (TBST) for 5 min each, the signals on the PVDF membrane were developed by incubating the membrane with chemiluminescence reagents; GADPH was used as the internal reference. The gray value of each target band was analyzed using ImageJ software.
4
Comprehensive Protein Expression Analysis
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Total protein was extracted from cells treated with EVO or DMSO by using the mammalian protein extraction reagent RIPA (Beyotime, China). The protein concentration was measured using BCA (bicinchoninic acid) protein assays (Beyotime, China). Protein samples were boiled with 6× SDS loading buffer for 5 min, separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked in 5% bovine serum albumin (BSA; Solarbio, Beijing, China) for 2h and then incubated with diluted specific primary antibodies overnight at 4°C. β-Actin was purchased from Zoonbio Technology (Beijing, China), while PCNA, β-catenin, c-Myc, cyclin D1, GSK-3β, Bcl-2, Bad, Bax, Caspase-3, cleaved Caspase-3, PARP, cleaved-PARP, MMP-2, MMP-7, MMP-9, vimentin, E-cadherin, N-cadherin and Snail were purchased from Cell Signaling Technology (Danvers, CO, USA). Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescent kit (Millipore, USA) according to the manufacturer’s protocol. The protein bands were pictured and analyzed by using the ChemiDoc MP Imaging System and Image Lab Software (Bio-Rad, California, USA).
5
Quantitative Western Blot Analysis of SGK3 Protein
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Total proteins were extracted from cells using RIPA lysis buffer (Kaiji Biotechnology Co., Ltd., Jiangsu, China). Protein concentrations were determined using protein assays (Beyotime Biotechnology Co., Ltd., Shanghai, China). Purified proteins (50 μL) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 2 h at room temperature in 5% non-fat milk and incubated overnight at 4 °C with an anti-SGK3 antibody (Abcam Company, 1:1000). Following incubation with a horseradish peroxidase (HRP), linked secondary antibody (Biosharp Biotechnology Co., Ltd., Hefei, China) (1:5000), the membranes were washed, and proteins were visualized using an enhanced chemiluminescence kit (PerkinElmer Inc., Waltham, MA, USA). Western blotting results were semi-quantitatively analyzed by Image-Pro Plus software.19
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