Figure 5.

Sedimentation gradient analysis of Xlrbpa and control construct MP2. (A) Oocyte lysates were fractionated on sucrose gradients and tested for the distribution of Xlrbpa (left). UV absorbance at 260 nm was recorded for all fractions (top) indicating the peak of 80S ribosomes in fractions 13–23. Representative fractions were tested by Western blotting with serum Rb7 for the presence of Xlrbpa (A). The majority of Xlrbpa could be found in the ribosomal peak fractions (fractions 16 and 18). In these fractions, degradation bands of lower molecular weight could also be detected. Minor amounts of Xlrbpa could be found at the top of the gradient (fraction 34), corresponding to free protein. However, the free protein was mostly degraded. Unfractionated oocyte extract was loaded in the first two lanes (Oo). Position of full-length Xlrbpa is indicated by an arrow. Ribosomal peaks from several primary gradients were concentrated in a Centricon microconcentration device (exclusion limit = 50 kD) and rerun on a second sucrose gradient (right). Supernatant (cen) and flowthrough (ft) from the microconcentration step was also monitored for the presence of Xlrbpa. Xlrbpa was exclusively present in the top chamber, indicating the presence of the protein in a particle larger than 50 kD. UV absorbance of the gradient rerun was monitored (top) and representative fractions were tested for the presence of Xlrbpa (A). Again, the majority of Xlrbpa could be found in the ribosomal peak fractions. No free protein could be found in the supernatant fraction (34). (B) Oocyte lysates of MP2-injected oocytes were fractionated on sucrose gradients. Fractions were monitored for the presence of MP2 protein by Western blotting with mAb 9E10 directed against the myc epitope. No MP2 protein could be found in the ribosomal peak fractions. Instead all MP2 remained at the top of the gradient. Unfractionated extracts of injected oocytes (Oo) were loaded as a control.