Figure 3.

Further Purification of ANN33/35.

Annexins were further purified using lipid binding (before) followed by size exclusion chromatography (after). The gels were either Colloidal Coomassie stained (C), silver stained (S), or used to perform protein gel blot analysis (W) to probe for ANN33/35 using anti-maize annexin antibody (Blackbourn et al., 1991). “Before” and “after” gels were run separately but used protein from the same preparation. The annexin doublet appeared on silver staining for 5 min, and the stains shown were developed for 15 min. Two separate preparations from size exclusion chromatography were used in an overdeveloped silver stain (40 min); samples are shown alongside a control for buffer only. This shows the presence of the p23 contaminant in the highly purified preparation. The sizes of ANN33/35 are 33 and 35 kD, respectively. All lanes were loaded with 3 μg protein.