Figure 5

Purification of [³⁵S]-biotinylated Pf BCCP-79. PfBCCP-79 was coexpressed with BirA and BioB in B834(DE3) cells in the presence of Na³⁵SO₄ and desthiobiotin, and purified by affinity chromatography. (A) Autoradiograph of a SDS-PAGE gel showing samples collected at each purification step. (B) Affinity blot analysis with streptavidin-HRP to detect biotinylated proteins. (C) Western blot analysis with PfBCCP antiserum. The gel samples are labeled as follows: flow through from metal chelate column (FT1); 30 mM imidazole wash (W1); 1 M NaCl wash (W2); 1% triton X-100 wash (W3); flow through from monomeric avidin column (FT2); buffer wash (W4); elution in 50 mM glycine pH 2.9 (E1).