RIPA lysis buffer (Invitrogen) containing PMSF (Bio-Rad, Shanghai, China) was used to collect proteins from SK-HEP1 and LM3 cells. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was applied in separating protein samples and a polyvinylidene fluoride membrane (PVDF) membrane (Invitrogen, Carlsbad, USA) was used to transfer the separated protein. The membrane was blocked in 5% skim milk at room temperature for 2 h in a shaker, and then incubated with the primary antibody at 4 °C overnight and subsequent incubation with the secondary antibody for 2 h. The blots were detected and imaged using the iBright FL1500 intelligent imaging system (Invitrogen, Carlsbad, USA). The details of antibodies in this study are listed in the supporting information .
Ibright fl1500 intelligent imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBright FL1500 intelligent imaging system is a versatile and advanced fluorescence imaging platform. It is designed to capture high-quality images and perform quantitative analysis of biological samples. The system integrates a range of features, including automated image acquisition, data analysis, and workflow management tools, to streamline the imaging process and facilitate reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
4 protocols using ibright fl1500 intelligent imaging system
1
Protein Extraction and Analysis from Liver Cancer Cells
2
Quantitative Protein Analysis of Liver Cancer
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The total protein of the two liver cancer cell lines after FTL shRNA transfection was extracted using RIPA lysis buffer (Invitrogen) containing PMSF (Bio–Rad, Shanghai, China), and the protein concentration was determined and quantified. The total protein was treated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF) (Invitrogen, Carlsbad, United States). After transfer, the membranes were blocked at room temperature for 2 h, then the primary antibody was added and incubated at 4°C overnight, and the secondary antibody was added and incubated at room temperature for 2 h. Finally, the iBright FL1500 intelligent imaging system (Invitrogen, Carlsbad, United States) was used to analyze the absorbance of the protein bands and calculate the relative protein expression level. The antibodies used in the study are listed in the Supplementary Material .
3
Cell Lysis and Protein Immunoblotting
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RIPA lysis buffer (Invitrogen, Carlsbad, USA) supplemented with 1% protease and phosphatase inhibitor (Thermo, Shanghai, China) was used to obtain protein from SK-HEP1 and HCCLM3 cells. The separation of protein samples with different molecular weights was achieved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Invitrogen), which was then blocked with 5% skimmed milk at room temperature for 90 minutes, incubated with the primary antibody at 4 °C overnight, and subsequently incubated with the HRP-conjugated secondary antibody for 2 hours at room temperature. The iBright FL1500 intelligent imaging system (Invitrogen) was employed to detect the signal and image the blot. The antibody information is listed in the supporting information.
4
Protein Extraction and Western Blot Analysis
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Proteins were obtained from SK-HEP1 and HCCLM3 cells using RIPA lysis buffer (Invitrogen, Carlsbad, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo, Shanghai, China). The separation of protein samples of different molecular weights was achieved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Separated proteins were electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen), blocked with 5% nonfat milk for 90 min at room temperature, and incubated with primary antibody overnight at 4 °C, followed by incubation with an HRP-conjugated secondary antibody. The combined secondary antibody was incubated for 2 h at room temperature. Signals were detected, and blots were imaged using an iBright FL1500 Intelligent Imaging System (Invitrogen).
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