Quantitative Protein Analysis using Western Blot

Western blot analyses of whole-cell protein lysates (RIPA lysis buffer supplemented with protease inhibitors, Thermo Fisher Scientific) and secreted proteins were performed as described [12 (link)]. Protein concentrations were assayed using a BCA protein assay Kit (Pierce, Rockfors, IL, USA). Equal amounts of protein were separated by SDS-PAGE using 4–20% gradient Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). After incubation with primary and secondary antibodies, the proteins were detected using Odyssey imaging (Li-Cor Biosciences). Relative band densities were quantified using Image Studio Lite Ver 5.2 software (Li-Cor Biosciences).

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Yin M., Tissari M., Tamminen J., Ylivinkka I., Rönty M., von Nandelstadh P., Lehti K., Hyytiäinen M., Myllärniemi M, & Koli K. (2017). Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma. Oncotarget, 8(58), 98280-98297.

Publication 2017

RIPA lysis buffer 4–20% gradient Tris-glycine gels nitrocellulose membranes Odyssey imaging Image Studio Lite Ver 5.2 software

Antibodies Assay Buffer Cell Gels Glycine Membranes Nitrocellulose Protease inhibitors Proteins Ripa Sds page Tris Western blot analyses

Corresponding Organization : University of Helsinki

Other organizations : Fimlab (Finland), Cancer Society of Finland, Karolinska Institutet, Helsinki University Hospital

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Relative band densities

control variables

Protein concentrations were assayed using a BCA protein assay Kit (Pierce, Rockfors, IL, USA)
Equal amounts of protein were separated by SDS-PAGE using 4–20% gradient Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad)

controls

Positive control: Not specified
Negative control: Not specified

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