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Fig. 5 | Plant Methods

Fig. 5

From: An improved protein lipid overlay assay for studying lipid–protein interactions

Fig. 5

PLO assays of the interaction between PS and the AHA2 C-terminus. a Analysis of PM H+-ATPase activity in vesicles of Arabidopsis Col-0. The indicated amount of PS or MeOH solvent (control) was added, and PM H+-ATPase activity was initiated by addition of 3 mM ATP. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP; 10 mM) was applied to collapse the pH gradient. b Comparison of PM H+-ATPase activities in a. c Interaction between PS and AHA2 C-terminus using CPLO and MPLO assays, respectively. PS was dissolved in 65:35:8 chloroform:MeOH:H2O and was spotted a PVDF membrane. The amount of PS spotted is shown at the top. The upper lane shows the interaction between PS and the AHA2 C-terminus using the CPLO assay. The lower lane shows the interaction between PS and the AHA2 C-terminus using the MPLO assay. The strips of CPLO and MPLO assays were performed exposure simultaneously using the same ECL reagent and the same settings. d Interaction between PS and AHA2 central loop using CPLO and MPLO assays, respectively. PS was dissolved in solvent 1 and spotted onto PVDF membrane. The amount of PS spotted on the membrane is shown at the top. The upper lane shows the interaction between PS and the AHA2 central loop using the CPLO assay. The lower lane shows the interaction between PS and the AHA2 central loop using the MPLO assay. The strips of CPLO and MPLO assays were performed exposure simultaneously using the same ECL reagent and the same settings. Data in b are mean ± standard deviation (SD) from five replicates. Student’s t-tests were used to analyze the statistical significance; significant differences (p ≤ 0.05) in b are indicated by different lower-case letters. The experiments in a and b were performed independently at least three times

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