RNA gels were performed as described (Skorupa et al., 2012 (link)). Briefly, 15 µg total RNA isolated with Trizol reagent, and the protocol was diluted in formamide loading buffer and denatured at 90°C for 5 min before loading onto a 15% TBE-Urea PAGE gel. Gels were electrophoresed at 200 V for 95 min at 4°C, stained with SYBR Gold (ThermoFisher Scientific), and imaged on an LAS 4000 Reader (Fujifilm). Northern blotting was performed as described (Kim et al., 2010 (link)). Briefly, following RNA gel electrophoresis, RNA was transferred to a Hybond+ nitrocellulose membrane (GE Lifesciences) at 10 V for 60 min at 4°C. RNA was UV-cross-linked in a Stratalinker (1200 mJ/cm2). Membranes were blocked in ultraHyb Oligo (ThermoFisher Scientific) for 30 min at 37°C. Dual digoxigenin-labelled DNA probes were added to the blocking solution (final concentration 1 nM) and incubated overnight at 37°C. Membranes were washed twice with low-stringency wash buffer (2× SSC, 0.1% SDS) and twice with high-stringency wash buffer (0.1× SSC, 0.1% SDS) at 37°C, then 2× SSC at room temperature. Membranes were processed using the DIG wash and block set according to instructions (Roche). CPD-Star Development Reagent (Roche) was added and images were acquired using a LAS 4000 Reader (Fujifilm). Dual digoxigenin-labelled probes are listed in Supplementary Table 2 .
Las 4000 reader
Manufactured by Fujifilm
The LAS 4000 Reader is a laboratory equipment designed for the analysis of electrophoresis gels and blots. It captures and digitizes images of these samples, providing accurate and reproducible data for research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Multi gauge software, by Fujifilm (2 mentions)
A5441, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Immobilon, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Roche (1 mentions)
Fusion capt software, by Vilber (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Ultrahyb oligo, by Thermo Fisher Scientific (1 mentions)
4 protocols using las 4000 reader
1
RNA Gel Electrophoresis and Northern Blotting
2
Western Blot Analysis of Angiopoietin
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Lysates were collected in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% sodium deoxycholate) with protease inhibitor (Sigma). Total protein was quantified using a macro BCA assay (ThermoFisher Scientific) and denatured for 10 min at 90°C in 1× laemmli buffer. Proteins were separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were incubated overnight in antibodies: goat anti-human Ang (1/500, AB-265-AN, R & D Systems, RRID: AB_354325) and mouse anti-actin (1/1000, A5441, Sigma, RRID: AB_476744). Membranes incubated for 2 h in HRP-conjugated secondary antibodies. ECL reagent was added (Immobilon, Merck), and images were taken on a LAS 4000 Reader (Fujifilm).
3
Immunoprecipitation and Western Blot Analysis
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These procedures were conducted as described in detail previously [13 (link)]. In brief, cells were lysed in normal lysis buffer (NLB: 50 mM Tris/HCl, pH 7.5; 1% Triton X-100; 137 mM sodium chloride; 1% glycerin; 1 mM sodium orthovanadate; 0.5 mM EDTA; 0.01 μg/μl leupeptin, 0.1 μg/μl aprotinin, 1 mM AEBSF). Blotted proteins were visualized with horseradish peroxidase-conjugated secondary antibodies (Roche) using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and either a LAS-4000 reader (FujiFilm) or a Fusion Solo chemiluminescence reader. Densitometry was performed using MultiGauge software (FujiFilm) or FusionCapt software (Vilber Lourmat). MS analysis of immunoprecipitated B-Raf complexes is described in detail in the expanded view.
4
HeLa Cell Lysis and Western Blot Analysis
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HeLa cells were grown in Dulbecco’s modified Eagle’s medium in a humidified atmosphere of 5% CO2 at 37°C. The media contained 10% fetal calf serum, penicillin (4 mM), and streptomycin (4 mM). For preparation of cell lysate, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in the indicated buffer for 5 min on ice. Afterward, the cell lysate was transferred into a 1.5-ml tube and cleared by centrifugation (10 min, 4°C, 20,000g). The supernatants were used for the following experiments. After separation of proteins by SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting, the proteins were detected by specific antibodies. Horseradish peroxidase–conjugated donkey anti-mouse immunoglobulin G (H&L) (610-703-124; Rockland) or goat anti-rabbit immunoglobulin G (H&L) (no. 7074, Cell Signaling) antibody was used as a secondary antibody. Antibody signals were detected by enhanced chemiluminescence using the LAS-4000 reader (Fujifilm) and quantified with MultiGauge software (FujiFilm).
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