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, 0.5% deoxycholate) or samples eluted from Anti‐FLAG M2 magnetic beads. For protein quantification, the Pierce Detergent Compatible Bradford Assay Reagent (Thermo Fisher Scientific) was used. All antibodies used in this study are listed in Dataset EV7 . Detection was performed with Western Lightning Plus‐ECL (PerkinElmer) or ECL Select Western Blotting Detection Reagent (Amersham) and the Vilber Fusion FX6 Edge imaging system (Vilber Lourmat). Protein levels were quantified in a semi‐automated manner using the ImageQuant TL 1D software with a rolling‐ball background correction. Signal intensities were normalized to the internal control (tubulin) before calculation of mean values. Quantification results are shown as data points and mean.
Pierce detergent compatible bradford assay reagent
Manufactured by Thermo Fisher Scientific
The Pierce Detergent Compatible Bradford Assay Reagent is a colorimetric assay solution designed to quantify the total protein concentration in samples that contain detergents. It is compatible with a wide range of detergents, including ionic, nonionic, and zwitterionic types. The reagent provides a linear, reproducible, and sensitive protein quantification method in the presence of detergents.
Automatically generated - may contain errors
Lab products found in correlation
Ecl select western blotting detection reagent, by Cytiva (2 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Select western blotting detection reagent, by GE Healthcare (1 mentions)
Fusion fx 6 edge system, by Vilber (1 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Sera mag carboxylate modified magnetic speedbeads, by GE Healthcare (1 mentions)
Trypsin lys c mix, by Promega (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Sonopuls mini20, by Bandelin (1 mentions)
5 protocols using pierce detergent compatible bradford assay reagent
1
SDS-PAGE and Immunoblot Analysis of Protein Samples
2
SDS-PAGE and Immunoblot Analysis
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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.
3
Isolation and Enrichment of Acetylated Mitochondrial Proteins
4
FLAG-tagged RNPS1 Mutant Expression
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Expression of FLAG-emGFP tagged RNPS1 mutants and FLAG-emGFP control was induced in stable cell lines (1.5 × 106 cells per 10 cm dish) using cumate (as described above) 72 h before cell lysis. The samples were lysed in 600 μl buffer E (20 mM HEPES–KOH (pH 7.9), 100 mM KCl, 10% glycerol, 1 mM DTT, Protease Inhibitor) in the presence of 1 μg ml–1 RNase A and sonicated using the Bandelin Sonopuls mini20 with 10 pulses (2.5 mm tip, 1s pulse, 50% amplitude). For immunoprecipitation, the protein concentration of the lysates was measured using Pierce Detergent Compatible Bradford Assay Reagent (Thermo Fisher Scientific) and adjusted in buffer E. Then, the lysates were loaded onto Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) and incubated for 2 h at 4°C with overhead shaking. After that, the beads were washed four times for 3 min with mild wash buffer (20 mM HEPES-KOH (pH 7.9), 137 mM NaCl, 2 mM MgCl2, 0.2% Triton X-100, 0.1% NP-40). For elution, 2 × 21.5 μl (42.5 μl total) of a 200 mg ml–1 dilution of FLAG peptides (Sigma) in 1× TBS was used.
5
Protein Analysis by SDS-PAGE and Immunoblot
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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, 0.5% deoxycholate) or samples eluted from Anti-FLAG M2 magnetic beads. For protein quantification, the Pierce Detergent Compatible Bradford Assay Reagent (Thermo Fisher Scientific) was used. All antibodies used in this study are listed in Dataset EV6.
Detection was performed with Western Lightning Plus-ECL (PerkinElmer) or ECL Select
Western Blotting Detection Reagent (Amersham) and the Vilber Fusion FX6 Edge imaging system (Vilber Lourmat).
Detection was performed with Western Lightning Plus-ECL (PerkinElmer) or ECL Select
Western Blotting Detection Reagent (Amersham) and the Vilber Fusion FX6 Edge imaging system (Vilber Lourmat).
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