Immunoblotting Analysis of Protein Samples

Cell samples were homogenized and solubilized in standard 2X Laemmli buffer (Invitrogen) supplemented with complete protease inhibitor cocktail (Roche). Following centrifugation at 20,000 g for 30 minutes, the concentration of supernatant proteins was analyzed using the Bio-Rad DC Protein Assay Kit according to the manufacturer's protocol. Extracted proteins were subjected to SDS-PAGE (Mini-Protean-III, Bio-Rad), stained with Coomassie or blotted onto PVDF membranes (Hybond-P, Amersham Bioscience), and probed with mouse monoclonal anti-Myc or anti-FLAG antibodies (Sigma). Molecular weight (MW) standards (MARK-12 and SeeBlue Plus2 from Invitrogen) were included on each gel. Equivalence of protein loading was confirmed by Amido Black staining of blots after immunodetection. Blocking, washing, incubation with diluted primary and secondary HRP-conjugated antibodies (Sigma), and visualization of immunodecorated bands by the Super-Signal West Pico PLUS chemiluminescent substrate (Thermo Scientific) were carried out as previously described [19 (link)].

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Torrado M., Maneiro E., Trujillo-Quintero J.P., Evangelista A., Mikhailov A.T, & Monserrat L. (2018). A Novel Heterozygous Intronic Mutation in the FBN1 Gene Contributes to FBN1 RNA Missplicing Events in the Marfan Syndrome. BioMed Research International, 2018, 3536495.

Publication 2018

complete protease inhibitor cocktail DC Protein Assay Kit Mini-Protean-III Hybond-P MARK-12 SeeBlue Plus2 HRP-conjugated antibodies Super-Signal West Pico PLUS chemiluminescent substrate

Amido black Anti antibodies Antibodies Assay Cell Centrifugation Laemmli buffer Membranes Mouse Protease inhibitor Protein Pvdf Sds page

Corresponding Organization : Universidade da Coruña

Other organizations : Vall d'Hebron Hospital Universitari

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Variable analysis

independent variables

Cell samples were homogenized and solubilized in standard 2X Laemmli buffer (Invitrogen) supplemented with complete protease inhibitor cocktail (Roche)

dependent variables

Concentration of supernatant proteins was analyzed using the Bio-Rad DC Protein Assay Kit
Extracted proteins were subjected to SDS-PAGE (Mini-Protean-III, Bio-Rad), stained with Coomassie or blotted onto PVDF membranes (Hybond-P, Amersham Bioscience), and probed with mouse monoclonal anti-Myc or anti-FLAG antibodies (Sigma)

control variables

Molecular weight (MW) standards (MARK-12 and SeeBlue Plus2 from Invitrogen) were included on each gel
Equivalence of protein loading was confirmed by Amido Black staining of blots after immunodetection

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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