Figure 4

Determination of protein spots detected by BEI sera on iRBC membrane protein extracts by 2-D immunoblotting. A 2-D immunoblot using a fluorescence-based method was performed to detect precisely and accurately antigenic protein spots. (A) Protein profile of iRBC membrane protein extracts pre-labelled with Cy5 and separated by 2-D electrophoresis. (B) Protein profile of the same protein sample used in "A" after Cy5 labelling, separation by 2-D electrophoresis and electroblotting onto nitrocellulose membrane. (C) Immunoblot pattern of a pool of 5 selected BEI sera revealed with a goat-anti-human IgG FITC-conjugate against iRBC membrane protein extracts. (D) Merged images of the electrobloted protein profile (e.i., "B") and the immunoblot pattern (e.i., "C"). Antigenic spots detected onto immunoblot (e.i., "C") are ringed and reported onto electrobloted protein profile (e.i., "B") and merged images (e.i., "D"). Antigenic spots which were excized for mass spectrometry (MS) analysis are marked with red or green circles onto gel image and those which were identified are indicated by Arabic numbers (e.i., "A"). Selected antigenic bands are indicated by Roman numbers and positioned according to their MW. Antigenic spots corresponding to selected antigenic bands are indicated by red circles, the others antigenic spots are assigned by green circles (A). A resume of the correspondence between Roman and Arabic numbers is indicated in additional file 2. MW: molecular weight, kDa: kiloDalton.