Panel A shows a step-by-step process for isolating yolk platelets (YPs) from Xenopus laevis oocytes. It includes photos of oocytes in a container, the separation of cytoplasm and YPs after centrifugation, and the final pellet containing intact YPs. Panel B displays an SDS-PAGE gel stained with Coomassie Blue and an immunoblot using an anti-lipovitellin (Lv) antibody, showing the total extracts (TE), and supernatants (S) and pellets (P) after consecutive washings and centrifugations. Molecular mass markers in kilodaltons (kDa) are on the left. Panel C shows immunofluorescence microscopy images of YPs where the yolk platelet membrane (YPM) is counterstained with fluorescent-conjugated wheat germ agglutinin (WGA; red), and the yolk proteins inside the YPs are labeled with a killifish anti-vitellogenin antibody that stains lipovitellin products (Lv, blue). The YPM is also labeled with vacuolar H+-ATPase (V-ATPase; green). Panel D shows similar immunofluorescence microscopy images where the YPM is labeled with protein disulfide isomerase (PDI; green). The purpose of combining these images is to illustrate the isolation process and the localization of specific proteins within the YPs.
Procedure for the isolation of intact YPs from X. laevis oocytes. (A) Oocytes (n = 30) are homogenized in 200 μl of Tris 80 mM (200 mOsm) at pH 7.5, by gentle pipetting, and the homogenate is centrifuged at 50 × g for 2 min at room temperature. The supernatant is discarded, and the pellet is washed twice in 500 μl of Tris 80 mM, pH 7.5, followed by 1-min centrifugation at 50 × g. Finally, the pellet is washed in 500 μl of Tris 80 mM, pH 7.5, and centrifuged for 1 min at 100 × g. On the right, a bright-field image of the final pellet containing intact YPs is shown. (B) SDS-PAGE gel stained with Coomassie blue and immunoblot using an α-Lv antibody of the TE, and supernatants (S) and pellets (P) after the consecutive washings and centrifugations. Molecular mass markers (kDa) are on the left. (C and D) Immunofluorescence microscopy images of YPs in which the YPM (arrowheads) is counterstained with fluorescent-conjugated WGA (red), whereas the yolk proteins inside the YPs are labeled with a killifish anti-vitellogenin antibody that stains Lv products (Lv, blue). The YPM is also labeled with vacuolar H+-ATPase (V-ATPase) or PDI (both in green), which are known components of this membrane (Fagotto and Maxfield, 1994; Jorgensen et al., 2009). Scale bars, 5 µm. TE, total extracts; Lv, lipovitellin. Source data are available for this figure: SourceData FS1.