MIF Western blot of serum proteins. A. Native serum MIF – Serum was diluted 1:20 in PBS and proteins were separated under native conditions then analyzed for MIF by Western blot analysis. Under native conditions, a broad band between 150 – 500 kDa was detected. B. Denatured and reduced serum MIF – Serum was diluted as in A and proteins separated under denaturing or reducing conditions. Denaturing conditions resulted in a single prominent band at 180 kDa and some minor bands between 80 and 120 kDa. Under reducing conditions, a single prominent 12-kDa band and a minor band at 24 kDa were observed. C. mAb Affinity purified serum MIF – MIF containing serum proteins were purified by sequential affinity chromatography through Protein G agarose, followed by MIF mAb affinity chromatography. Nonsticking proteins as well as elution fractions were separated by NuPAGE gel electrophoresis under denaturing or reducing conditions, transferred to PVDF and MIF containing bands identified using a polyclonal antibody (R&D Systems, 1:1000 dilution). Lanes left to right: NS Protein G – non-sticking fraction from Protein G column; NS MIF mAb – non-sticking fraction from MIF monoclonal antibody column; Elution – third elution fraction; Elution conc – third elution fraction concentrated 800 fold; MIF standard – 7.5 ng of recombinant MIF protein. Numbers and lines to the left of each Western blot indicate location of molecular weight markers.