Analytical SEC of Protein Complexes

Analytical SEC experiments were performed as previously described (9 (link), 32 (link)). Briefly, either a Superose 6 PC 3.2/300 column (GE Healthcare) for in vitro translated p63 or a Superose 6 GL 10/300 column (GE Healthcare) for cell lysates of transiently transfected H1299 cells were used. H1299 cell lysate was prepared by three cycles of freezing and thawing in SEC running buffer (50 mM Tris pH 7.5; 150 mM NaCl; 1 mM DTT) with protease inhibitors and Benzonase followed by removal of cell debris by centrifugation. Before injection, cell lysates were incubated at 37 °C for 15 min. The Superose 6 GL 10/300 column was calibrated using the Gel Filtration HMW and LMW Calibration Kits.

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Russo C., Osterburg C., Sirico A., Antonini D., Ambrosio R., Würz J.M., Rinnenthal J., Ferniani M., Kehrloesser S., Schäfer B., Güntert P., Sinha S., Dötsch V, & Missero C. (2018). Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome. Proceedings of the National Academy of Sciences of the United States of America, 115(5), E906-E915.

Publication 2018

Superose 6 PC 3.2/300 column Superose 6 GL 10/300 column

Benzonase Buffer Cell Centrifugation Gel filtration Nacl Protease inhibitors Tris

Corresponding Organization :

Other organizations : Tecnologie Avanzate (Italy), Goethe University Frankfurt, University of Naples Federico II, SDN Istituto di Ricerca Diagnostica e Nucleare, Istituti di Ricovero e Cura a Carattere Scientifico, University at Buffalo, State University of New York

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Variable analysis

independent variables

Type of column used for SEC experiments (Superose 6 PC 3.2/300 column for in vitro translated p63 or Superose 6 GL 10/300 column for cell lysates of transiently transfected H1299 cells)

dependent variables

Elution profile/size-exclusion chromatography profile of in vitro translated p63 or cell lysates of transiently transfected H1299 cells

control variables

SEC running buffer (50 mM Tris pH 7.5; 150 mM NaCl; 1 mM DTT) with protease inhibitors and Benzonase
Incubation of cell lysates at 37 °C for 15 min before injection
Calibration of the Superose 6 GL 10/300 column using the Gel Filtration HMW and LMW Calibration Kits

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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