Journal: Molecular and biochemical parasitology
Article Title: The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1
Figure Lengend Snippet: PF3D7_0402000 (D90c) interacts with ANK1 and 4.1R in co-precipitation assays A) and C) Expression of proteins tagged with C-terminal fragment of firefly luciferase protein (C-FLuc) and 3XFLAG. ANK1 FG1 and 4.1R FG1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of erythrocyte ankyrin and 4.1R with MBP-His-tagged PF3D7_0402000 (MBP-D90c-His). C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE analysis and western blotting using anti-FLAG antibody (top panel) and anti-MBP antibody (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left. D) Co-affinity purification of erythrocyte ankyrin and 4.1R with GST tagged-PF3D7_0402000 (GST-D90c). GST and GST-D90c were in vitro translated in WGE, incubated with C-FLuc-3XFLAG-tagged proteins from (C) and purified with GST beads. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti-FLAG (top panel) and anti-GST antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.
Article Snippet: GST or GST-D90c were mixed with 3XFLAG-C-FLuc tagged ANK1 and 4.1R, incubated with GST beads (Thermo Scientific) overnight at 4° C in presence of protease inhibitor (PI) cocktail (Roche), washed three times with PBS+0.1% TritonX-100, eluted by boiling in Laemmli SDS-PAGE sample loading buffer, and subjected to western blot analysis.
Techniques: Expressing, Luciferase, In Vitro, Western Blot, FLAG-tag, Molecular Weight, Affinity Purification, Incubation, Purification, SDS Page