Proteins were extracted from the HUVECs of the different cultures by cell lysis buffer (Cell Signaling Technology, Danvers, MA). Equal amounts of protein were separated by 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore Corporation). The PVDF membranes were blocked in 5% skim milk at room temperature for 1 h and then incubated with the corresponding primary antibody (1:1000) at room temperature for 2 h. The primary antibodies were monoclonal antibodies against β-actin (ProteinTech, Wuhan, China), PTEN (ProteinTech, Wuhan, China), SET8 (ProteinTech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), forkhead box protein O1 (FOXO1) (Cell Signaling Technology, Danvers, MA), and e-selectin (Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal antibodies against ICAM-1 (Cell Signaling Technology, Danvers, MA) and p-p65 (Cell Signaling Technology, Danvers, MA). The membranes were incubated with the corresponding secondary antibody (1:1000) at room temperature for 1 h. The membranes were then washed, and the proteins were detected by a LAS-4000 mini CCD camera (GE Healthcare).
Las 4000 mini ccd camera
Manufactured by GE Healthcare
Sourced in United States
The Las-4000 mini CCD camera is a compact, high-performance imaging device designed for laboratory applications. It features a charge-coupled device (CCD) sensor that captures and digitizes images for data analysis and documentation purposes. The camera provides reliable image capture and transfer capabilities, enabling users to effectively document and analyze their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (5 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Icam 1, by Cell Signaling Technology (1 mentions)
H4k20me1, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
E selectin, by Santa Cruz Biotechnology (1 mentions)
Forkhead box protein o1 foxo1, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Ready gel, by Bio-Rad (1 mentions)
Complete protein inhibitor cocktail, by Roche (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using las 4000 mini ccd camera
1
Protein Expression Analysis in HUVEC Cells
2
Protein Detection by Western Blot
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The target proteins in cells were detected by western blot analysis. Equal amounts of proteins achieved from cells were separated by 10% SDS-polyacrylamide gels and immobilized on polyvinylidene fluoride membranes (Millipore, Billeruca, MA) using a full-wet electroblotting system. The membranes were blocked with 5% fat-free milk in PBST for 1 hour, then hybridized with 1,000-1 dilution of specific primary antibodies overnight at 4 °C. The membranes were washed with PBST, and then incubated with a 1,500-1 dilution of Goat anti-rabbit IgG, HRP conjugate (Proteintech, Wuhan, China) for 1 hour at room temperature. After washing, the signals were visible after development with a chemiluminescence (ECL) reagent (Millipore Corporation, USA) and detected using a LAS-4000 mini CCD camera (GE Healthcare).
3
Quantification of FGF-1 and FGF-3 Levels
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The conditioned media of fibroblasts and colon cancer cells were collected and centrifuged to obtain supernatants. FGF-1 levels were determined using a specific ELISA kit against FGF-1 (Elabscience, Wuhan, China) according to the manufacturer’s instructions. The data are expressed as the target molecule (pg) per total media (mL) for each sample. To detect FGF-3, the cell lysates were analyzed by immunoblotting using an anti-FGF-3 primary antibody. The signals were detected using a Las-4000 mini CCD camera (GE Healthcare, Marlborough, USA).
4
Colon Tissue Protein Extraction and Analysis
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Colon tissues were homogenised and sonicated in RIPA lysis buffer (Beyotime Biotechnology, Suzhou, China) supplemented with protease and phosphatase inhibitors (Roche, Rotkreuz, Switzerland) to detect phospho-tyrosine, phospho-Erk, total Erk, phospho-Mek, total Mek, MMP7, and FGFR4. In some experiments, the supernatants of cell lysates were incubated with 2 μg of rabbit anti-FGFR4 antibody and then precipitated with 20 μL of protein G Sepharose 4 Fast Flow (GE Healthcare) to detect the level of tyrosine phosphorylation with an anti-phospho-tyrosine antibody. To detect activated MMP-7, the proteins precipitated from conditioned medium using 10% trichloroacetic acid were analyzed using an anti-MMP7 primary antibody. The signals were detected using a Las-4000 mini CCD camera (GE Healthcare).
5
Western Blot Analysis of MAFK Protein
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Forty eight hours after transfection, the cells were harvested and solubilized with lysis buffer with complete protein inhibitor cocktail (Roche). Protein concentrations were determined using the BCA Protein kit (Thermo Fisher Scientific). Thirty milligrams protein was size-fractionated using 10% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis Ready-Gels (Bio-Rad Laboratories) before electroblotting onto nitrocellulose membranes. Membranes were then blocked with 5% skim milk in 0.1% Tween 20 (Sigma-Aldrich) Tris-buffered saline (TBST). MAFK and GAPDH primary antibodies were diluted (1:1000) in TBST with 5% milk prior to use. Primary antibody incubations were then performed overnight at 4°C after antigen blocking. Thereafter, membranes were washed with TBST and incubated in the corresponding horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h. The blotted membranes were treated using the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology Inc.), and signals were detected using a Las-4000 mini CCD camera (GE Healthcare). GAPDH was used as a loading control.
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