Fig. 1.

Gel exclusion size chromatography of human serum following the removal of albumin and immunoglobulin. A) Serum following the removal of IgG and albumin was size fractionated on a HiPrep Sephacryl 16/60 S-300 column under native conditions resulting in five peaks. OD280 determined using Monitore UPC-900 flow through cell and the ÄKTA fast pressure liquid chromatography system (GE Healthcare, Piscataway, NJ). B) Sephacryl fractions corresponding to each peak were pooled, concentrated to a final volume of 1.5 ml and 15 µl aliquots analyzed by SDS-PAGE and Coomassie stained for total protein (left panel). Coomassie stained gels were imaged using the 700 nm channel of the Odyssey infrared imager (LI-COR). Duplicate gels were prepared and analyzed by Western blotting for MIF (right panel). MIF biotinylated detection antibody and strepavidin 800 CW complexes binding to immobilized MIF complexes on the blots were detected using the 800 nm channel of the Odyssey infrared imager. Fraction 5 was chosen for further analysis since the fraction had a single intense MIF containing band on the Western blot (right panel) and only three main protein bands (approximately 62 kDa) detected by Coomassie staining (left panel). Lane 1–5, fractions 1–5 respectively.