Figure 3

Purification of recombinant APC proteins by a two-step method using IMAC and APC-NT mAb affinity chromatography. A) Schematic diagram depicting the purification scheme used to purify recombinant APC proteins. B) Purification of recombinant APC proteins. Sf9 cells (2×10⁹) infected with either fl-APC, APC(1–1638) or APC(1–1311) were and purified using Ni-NTA resin. Proteins were eluted using a stepwise gradient of 20, 50, 100 and 250 mM imidazole. 1% of each fraction was resolved using 4-12% Bis-Tris SDS-PAGE and stained with Coomassie blue (Left panel, 1 = 20 mM, 2 = 50 mM, 3 = 100 mM, 4 = 250 mM). Pooled fractions containing APC were applied to a 5 ml APC-NT mAb column and eluted with 0.1 M Glycine pH 3 in 5× 2 ml fractions (right). 1% of each fraction was resolved as above. The expected sizes of APC proteins are indicated by arrows and relative positions of molecular weight markers in kDa are indicated. C) Purified recombinant APC proteins. APC proteins were purified using the optimized strategy (A). The amount of APC was estimated by comparison to a band representing 1 μg BSA. The expected size of APC proteins are indicated by arrows and relative positions of molecular weight markers are indicated. Purified APC bands were excised and analysed using LC-MS/MS. MASCOT Protein score [26], peptide number, and amino acid coverage are indicated for each protein.