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Dataset for graphs for Vps1 lipid binding (Smaczynska et al 2019)

posted on 2019-04-02, 13:08 authored by Kathryn Ayscough, Iwona Smaczynska-de Rooij, Ellen Allwood, Sarah Palmer, Christopher Marklew
Dataset files for Figures 2,3,6 graphs in 2019 PLoS One manuscript

Preparation of liposomes. 22 µl of a 25 mg/ml solution of Folch fraction-1 (Sigma) or a purpose made mixture of 65% PC, 15% PE, and either 20% PI(4)P, PI(3)P or PI(4,5)P2 (Avanti Polar Lipids dissolved in chloroform) was prepared, and dried under a nitrogen stream. Liposomes were formed by resuspension of the dried lipids in 200 µl F-buffer (0.2 mM CaCl2, 12 mM Tris/HCl, pH 8.0, 1 mM NaN3, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA) at 60°C for 30 mins with regular agitation.

Liposome cosedimentation assays. Purified Vps1 was pre-spun at 350,000g for 15 mins (Beckman Ultra centrifuge, TL100 rotor). Pre-spun protein was immediately added to 0.22 mg/ml liposomes to a concentration of 0.4 µM, in F-buffer, (final volume 50 µl). For full length Vps1 the protein-liposome mixture was immediately re-spun at 110,000g for 15 mins. For Insert B the protein-liposome mixture was incubated for 30 mins at room temperature, before pelleting the liposomes at 280,000g for 15 mins.

After centrifugation supernatants and pellets were separated, and pellets resuspended in 50 µl of F-buffer. Strataclean resin (Stratagene) was then used to concentrate the protein in each sample. 10 µl of Strataclean resin was added to, and briefly mixed with, each sample, before being pelleted by spinning at 8000g in a bench top micro-centrifuge for 2 mins. The supernatant was removed and the pellet resuspended in 10 µl of SDS-PAGE loading buffer. Protein was visualised by SDS-PAGE and Coomassie staining. Data was analysed by densitometry using ImageLab software (BioRad).




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