Figure 5.

Longer incubation of leaf samples with biotin reveals new candidate phytaspase interactors. After prolonged (16 h) incubation of leaf samples with 200 μM biotin, proteins were extracted from the intracellular fractions of NtPhyt-TurboID- or SP-TurboID-producing leaves (Phyt and SP, respectively; obtained from equal 5 g amounts of leaf material) in the presence of 0.5% dodecyl maltoside. Biotinylated proteins that precipitated within the 50–70% interval of ammonium sulphate saturation were further affinity-purified using streptavidin magnetic beads. The eluted protein samples were concentrated by acetone precipitation, dissolved in 45 μL of SDS-containing buffer and analysed by SDS electrophoresis in a 6–16% gradient polyacrylamide gel. Proteins were visualized after Coomassie Blue staining. M, molecular weights of the protein markers. Arrows with numbers indicate the bands chosen for subsequent protein identification.