Proteins were extracted as described in Stanisz et al (2014 ). Briefly, 25 μg (unless otherwise specified) of protein were separated by 10% SDS–polyacrylamide gel electrophoresis and were transferred onto a nitrocellulose membrane (Amersham Protran Premium 0.45 μm; GE Healthcare #10600003). After transfer, membranes were blocked in 5% bovine serum albumin prior to primary antibody incubation overnight. Antibodies and used dilutions are listed in Appendix Table S7 . Fluorescent secondary antibodies (IRDye; LI‐COR, Lincoln, USA) were incubated for 1 h in the dark, at room temperature. Membranes were scanned and quantified using an Odyssey Sa Infrared imaging system (LI‐COR, USA).
Amersham protran premium 0.45 μm
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham Protran Premium 0.45 μm is a nitrocellulose membrane designed for protein transfer and immobilization in western blotting applications. It has a pore size of 0.45 micrometers.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey sa infrared imaging system, by LI COR (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
2 protocols using amersham protran premium 0.45 μm
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 1% NP-40, 1 mM Na3 VO4, protease inhibitor cocktail; Sigma Aldrich), subjected to SDS-PAGE and blotted onto Amersham Protran Premium 0.45 μM nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, UK). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Primary antibodies were purchased from Cell Signaling Technology (Leiden, The Netherlands). Blots were developed using horseradish peroxidase-conjugated secondary antibody (Dako, Jena, Germany) at room temperature for 1 h and the Amersham ECL prime detection reagent (GE Healthcare), in accordance to manufacturers protocol. Chemiluminescence signals were detected with the ChemiDocTouch Imaging System (BioRad) and images were processed with the ImageLab 5.2 Software (BioRad). HDAC4 detection signals were normalized to the β-actin signal, for CDK4 the ratio between phosphorylated and non-phosphorylated protein was calculated (n = 4).
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