10.15131/shef.data.7932062.v1
Kathryn Ayscough
Kathryn
Ayscough
Iwona Smaczynska-de Rooij
Iwona
Smaczynska-de Rooij
Ellen Allwood
Ellen
Allwood
Sarah Palmer
Sarah
Palmer
Christopher Marklew
Christopher
Marklew
Dataset for graphs for Vps1 lipid binding (Smaczynska et al 2019)
The University of Sheffield
2019
Vps1
lipid
dynamin
yeast
endocytosis
Cell Biology
2019-04-02 13:08:57
Dataset
https://orda.shef.ac.uk/articles/dataset/Dataset_for_graphs_for_Vps1_lipid_binding_Smaczynska_et_al_2019_/7932062
<div>Dataset files for Figures 2,3,6 graphs in 2019 PLoS One manuscript</div><div>
<p><a></a><b>Preparation of liposomes. </b>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)P<sub>2</sub> (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.</p>
<p><b>Liposome cosedimentation
assays. </b>Purified
Vps1 was pre-spun at 350,000<i>g</i> 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 110,000<i>g</i> for 15 mins. For
Insert B the protein-liposome mixture was incubated for 30 mins at room
temperature, before pelleting the liposomes at 280,000<i>g</i> for 15 mins.</p>
<p>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 8000<i>g</i>
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).</p>
<p> </p>
</div><div>
<p><a></a><b></b></p><p> </p>
</div><div><br></div>