Sucrose Gradient Fractionation of Digested Samples

The digested samples were then loaded on top of previously prepared sucrose gradients (gradient 5% to 50% sucrose in 5% steps and 1 mL volume per step) and separated by ultracentrifugation (Sorvall WX+ Ultracentrifuge from Thermo Scientific with a SW41 Ti-Rotor) overnight (18 h, 110,000–115,000× g, 4 °C). The gradient was fractionated in 395 µL aliquots (25 fractions per gradient). The samples were snap-frozen in liquid nitrogen and stored at −80 °C for later use for Western blot validation or mass spectrometry analysis. For a detailed protocol, refer to [30 (link)].

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Rajagopal V., Loubal A.S., Engel N., Wassmer E., Seiler J., Schilling O., Caudron-Herger M, & Diederichs S. (2022). Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells. Cancers, 14(24), 6109.

Publication 2022

Sorvall WX+ Ultracentrifuge

Frozen Mass spectrometry Nitrogen Sucrose Ultracentrifugation Western blot

Corresponding Organization : University of Freiburg

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Variable analysis

independent variables

Sucrose gradient concentration (5% to 50% in 5% steps)

dependent variables

Protein separation by ultracentrifugation

control variables

Ultracentrifugation conditions (110,000–115,000× g, 18 h, 4 °C)
Sucrose gradient volume (1 mL per step)
Fractionation volume (395 µL per fraction)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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