Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome
J. Clin. Invest. Tamara Manuelian, et al. 111:1181 doi:10.1172/JCI16651 [Go to this article.]

Figure 5
Heparin affinity chromatography binding of recombinant wild-type deletion mutant factor H (SCRs 8-20). (a) Culture supernatant of insect cells infected with recombinant baculovirus coding for the recombinant wild-type protein (FH 8-20) and the two mutant forms, R1210C mutant (i.e., FH 8-20/R1210C) or the R1215G mutant (i.e., FH 8-20/R1215G) was applied to heparin affinity chromatography. After loading, the column was thoroughly washed, and bound proteins were eluted by an NaCl gradient. Absorbencies are indicated for recombinant wild-type protein by the dashed line, the R1210C mutant by the solid line, and the R1215 mutant by the dotted line. The identical conductivity graphs show that elution was performed under identical conditions. mAu, milliampere units. (b) SDS-PAGE and Western blot analysis of fractions 34–41 of the individual proteins. (c) The separation yields pure protein as shown for wild-type protein fractions 34–41 after SDS-PAGE separation in combination with silver staining. The mobility of the marker proteins is indicated on the left.