SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed using protein samples harvested with RIPA buffer (50 mM Tris/HCl pH 8.0, 0.1% SDS, 150 mM NaCl, 1% IGEPAL CA 630, 0.5% deoxycholate) or samples eluted from Anti-FLAG M2 magnetic beads (Sigma-Aldrich). For protein quantification, the Pierce Detergent Compatible Bradford Assay Reagent (Thermo Fisher Scientific) was used. All antibodies were used at the indicated dilutions in 50 mM Tris [pH 7.2], 150 mM NaCl with 0.2% Tween-20, and 5% skim milk powder. Amersham ECL Prime or Select Western Blotting Detection Reagent (GE Healthcare) in combination with the Fusion FX-6 Edge system (Vilber Lourmat) was used for visualization. All antibodies used in this study are listed in Supplementary Data 6 . Protein bands detected with the Fusion FX-6 Edge system (Vilber Lourmat) using the Evolution-Capt Edge software (version 18.05) were quantified in a semi-automated manner using the ImageQuant TL 1D software (version 8.1) with a rolling-ball background correction. The control condition was set to unity, quantification results are shown as data points and mean.
Fusion fx 6 edge system
Manufactured by Vilber
Sourced in United States, France
The Fusion FX-6 Edge system is a laboratory instrument designed for imaging and analysis. It features a high-resolution camera and advanced software for capturing and processing images of various samples.
Automatically generated - may contain errors
Lab products found in correlation
Evolutioncapt v18, by Vilber (2 mentions)
Coreldraw 2020, by Vilber (2 mentions)
Select western blotting detection reagent, by GE Healthcare (1 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Pierce detergent compatible bradford assay reagent, by Thermo Fisher Scientific (1 mentions)
Imagequant tl 1d software, by GE Healthcare (1 mentions)
Amersham ecl prime or select western blotting detection reagent, by GE Healthcare (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using fusion fx 6 edge system
1
SDS-PAGE and Immunoblot Analysis
2
Immunoblotting Analysis of Protein Complexes
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Protein samples from co-immunoprecipitation were loaded onto SDS-polyacrylamide gels using SDS-sample buffer, separated by gel-electrophoresis and analysed by immunoblotting. All antibodies were diluted in 50 mM Tris [pH 7.2], 150 mM NaCl with 0.2% Tween-20 and 5% skim milk powder. Antibodies and dilutions are listed in Supplementary Table S1 . For visualization, we used Amersham ECL Prime or Select Western Blotting Detection Reagent (GE Healthcare) in combination with the Fusion FX-6 Edge system (Vilber Lourmat). Quantification was performed in a semi-automated manner using the ImageQuant TL 1D software (GE Healthcare Life Sciences) with a rolling-ball background correction.
3
GFP Protein Detection in N2a Cells
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Transfected N2a cells were lysed with ice-cold Lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 10% glycerol) supplemented with a protease inhibitor cocktail (Roche). Extracts were cleared by centrifugation at 15,000 × g and 4 °C for 15 min, boiled at 95 °C for 5 min and finally analyzed by SDS-PAGE and Western blots using antibodies against GFP, α-tubulin as loading control, and HA-tag when required. HRP-conjugated secondary antibodies were used for enhanced chemiluminescence (ECL) detection in a Fusion FX6 Edge system (Vilber). Quantification of all blots was done using ImageJ v1.53c, and images were edited using EvolutionCapt v18.11 (Vilber) and CorelDRAW 2020.
4
Quantitative Protein Analysis via Western Blot
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To determine potential changes in protein levels, Western blot analysis of selected proteins was carried out. The proteins were isolated in 100 μl of a urea buffer (8 M, pH 8.5) combined with lysis using a TissueLyser II (Qiagen, Hilden, Germany) operated for 5 min at 20 Hz. The cell residues were separated by centrifugation at a relative centrifugal force (rcf) of 10,000 for 10 min. Total protein concentration was evaluated using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Darmstadt, Germany). In general, 20 μg of total protein was then subjected to SDS-PAGE and transferred to nitrocellulose membranes following standard protocols. Primary antibodies against CYP1A1 (ABIN1872160; Antibodies-online Inc., Limerick, PA, USA), CYP1B1 (ABIN3184162), XPC (ABIN2855495), XPG (ABIN3187504), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABIN2666338) were used for subsequent immunostaining, followed by visualization with appropriate horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and enhanced chemiluminescence (34078; Thermo Scientific, Waltham, MA, USA) for detection in the Fusion FX6 Edge system (Vilber Lourmat, Eberhardzell, Germany).
5
Quantification of PlyAZ3a^T in CSF and Serum
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The amount of PlyAZ3aT in CSF and serum samples was assessed by quantitative near infrared Western Blot (NIR-WB). Proteins in CSF or serum were separated using 10% SDS-polyacrylamide gels. Western blotting and immunostaining were performed with WesternBrightTM MCF and MCF-IR kit (Advansta, San Jose, USA) according to manufacturer’s instructions. PlyAZ3aT was detected as a 41 kD band, by using a primary antibody (custom-produced PlyAZ3aT-specific polyclonal rabbit antibody, David’s Biotechnology, Regensburg, Germany). Fluorescence was measured using a Fusion FX6 EDGE System (Vilber Lourmat, Collégien, France) with an exposure time of 2 min. Quantification of PlyAZ3aT was performed by comparison to a standard curve of PlyAZ3aT ranging from 2–0.02 μg using FIJI (version 1.8). The limit of detection was 10 μg/ml.
6
Transfected N2a Cell Lysis and Immunoblotting
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Transfected N2a cells were lysed with ice-cold Lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 10% glycerol) supplemented with a protease inhibitor cocktail (Roche). Extracts were cleared by centrifugation at 15,000xg and 4°C, boiled at 95°C for 5 min and finally analyzed by SDS-PAGE and Western blots using antibodies against GFP, α-tubulin as loading control, and HAtag when required. HRP-conjugated secondary antibodies were used for enhanced chemiluminescence (ECL) detection in a Fusion FX6 Edge system (Vilber). Quantification of all blots was done using ImageJ v1.53c, and images were edited using EvolutionCapt v18.11 (Vilber) and CorelDRAW 2020.
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