Fig. 2.

Isolation of MIF containing protein complexes in Sephacryl fraction 5. Fraction 5 was chosen for further analysis since this fraction had a single intense MIF containing band as determined by Western blot. A) Proteins were separated by 2D-gel electrophoresis (IEF linear gradient pH 3 – 10) followed by SDS-PAGE (4–12% Bis-Tris gel) and Coomassie staining. Coomassie staining bands were imaged using the 700 nm channel of the Odyssey infrared imager. B) Duplicate gels were prepared for MIF Western blotting. Images were obtained using strepavidin conjugated 800 CW infrared dye and the Odyssey imager. The matched region on the Coomassie stained gel (arrows) was excised and subjected to mass spec analysis (Hybrid quadrupole-time of flight, ABI QSTAR XL), which identified the MIF protein as Serpinc1 also known as anti-thrombin III (ATIII, See Table 1).