Protran 0.45 μm nitrocellulose membrane

The preparation of oocyte membranes and the semi-quantification of PfCRT protein was carried out using a protocol described in detail elsewhere [42 (link)]. Protein samples prepared from oocyte membranes were separated on a 4–12% Bis-Tris SDS-polyacrylamide gel (Life Technologies) and transferred to a Protran 0.45 μM nitrocellulose membrane (Amersham, GE Healthcare Life Sciences). The membranes were probed with rabbit anti-PfCRT antibody (concentration of 1:4,000; Genscript) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (1:8,000; Life Technologies, cat. no. 656120). Validation of the specificity of the anti-PfCRT antibody has been published in detail elsewhere [42 (link)]. The PfCRT band for each variant was detected by chemiluminescence (Pierce), quantified using the Image J software [75 (link)], and expressed as a percentage of the intensity measured for the PfCRT^K1 band. Total protein staining was used to evaluate sample loading and efficiency of transfer as outlined previously [42 (link)]. Between five and seven independent experiments were performed (on oocytes from different frogs), and in each experiment measurements were averaged from two independent replicates.

Proteins were separated by 10% SDS-PAGE and transferred to a Protran 0.45 μm nitrocellulose membrane (GE Healthcare) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol). The membrane was washed with PBST (PBS supplemented with 0.05% Tween) and blocked with 10% low fat milk/PBST. All antibodies were diluted in 1% low fat milk/PBST. AffiniPure goat secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch) were used. The membrane was incubated with SuperSignal West Femto/Pico Substrate (Thermo Fisher Scientific) and the image was developed using LAS-3000 Imager (Fujifilm).