Proteinpurification,Westernblotanalysisandinhibition.ppt

Protein purification, Western blot analysis and inhibition ELISA A Recombinant proteins 5?μg were separated by SDS/PAGE and were stained with Coomassie Blue. Andreas?G. Glaser et al. Biochem. J. 2006;396:41-49 ?2006 by Portland Press Ltd Protein purification, Western blot analysis and inhibition ELISA A Recombinant proteins 5?μg were separated by SDS/PAGE and were stained with Coomassie Blue. Molecular-mass standards in kDa are indicated in lane 1. Lane 2, Mala s 6; lane 3, Asp f 27; lane 4, Asp f 11; lane 5, human CyP B. B Specific IgE binding of recombinant CyPs analysed by Western blotting using serum pools from individuals sensitized to M. sympodialis left and A. fumigatus right . Lanes 1 and 5, Mala s 6; lanes 2 and 6, Asp f 27; lanes 3 and 7, Asp f 11; lanes 4 and 8, human CyP B. Molecular-mass sizes are indicated in kDa. C Competitive inhibition of IgE binding to recombinant human CyP B coated on a solid phase. Pooled serum from A. fumigatus-sensitized patients was pre-incubated with increasing amounts of recombinant human CyP B ▲ , Mala s 6 ● , Asp f 27 □ , Asp f 11 ■ or BSA as negative control ◆ . Pre-incubated serum samples were transferred to wells coated with human CyP B, and IgE bound was analysed by ELISA.