Size-Exclusion Chromatography of Protein Complexes

Analytical SEC was performed in phosphate buffer (50 mM Tris pH 7.5, 150 mM NaCl, 20 mM CHAPS, 1 mM DTT) at 4 °C using a Superose 3.2/300 column (GE Healthcare) (injection volume 50 μl; flow rate 50 μl/min; fraction size 50 μl). SEC fractions were quantified by western blotting. Analytical SEC of TDs was performed in KPKCl buffer at 4 °C using a Superdex 10/300 column (GE Healthcare) [32 (link)].

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Raab M., Matthess Y., Raab C.A., Gutfreund N., Dötsch V., Becker S., Sanhaji M, & Strebhardt K. (2021). A dimerization-dependent mechanism regulates enzymatic activation and nuclear entry of PLK1. Oncogene, 41(3), 372-386.

Publication 2021

Superose 3.2/300 column Superdex 10/300 column

Buffer Chaps Nacl Phosphate Tris

Corresponding Organization : Heidelberg University

Other organizations : Goethe University Frankfurt

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Variable analysis

independent variables

Buffer composition (50 mM Tris pH 7.5, 150 mM NaCl, 20 mM CHAPS, 1 mM DTT)
Column type (Superose 3.2/300 for analytical SEC, Superdex 10/300 for TDs)
Injection volume (50 μl)
Flow rate (50 μl/min)
Fraction size (50 μl)

dependent variables

Protein quantification in SEC fractions (measured by western blotting)

control variables

Temperature (4 °C)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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