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Global assessment of protein-protein crosslinking in ex vivo eRIC samples

ABSTRACT: Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Kidneys were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC (PMID: 30352994). The proteins captured by eRIC were separated by gel electrophoresis, divided into 7 fractions of defined mass, and identified by MS to determine their distribution across the fractions. Crosslinking to other proteins should shift the signal to higher mass fractions. The analyses were conducted in triplicate.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Kidney

SUBMITTER: Frank Stein

LAB HEAD: Matthias W. Hentze

PROVIDER: PXD038100 | Pride | 2023-03-02

REPOSITORIES: Pride

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