Figure 5

Three examples of confirmation of antigen identification: PGAM1, syntenin and agrin by duplex Western blotting. Spots number 30 and 31 were both identified as PGAM1 by mass spectrometry. 2-D Western blots were performed with seven individual human sera and detected with a secondary antibody conjugated with the fluorescent dye IRDye 800 as described before. The blots were then reprobed with a PGAM1-specific antiserum that was detected with a secondary antibody coupled with Alexa Fluor 680, confirming the identity of spots 30 and 31 as PGAM1 in every case. Three examples for the confirmation of PGAM1 as the antigen number 31 are shown (a). Spot number 41 was reactive with 8/21 CRC and with 5/24 control sera. All positive sera were used in duplex Western blots with a syntenin-specific antiserum, confirming the correct assignment of immunoreactions for all but one (CRC) serum. Representative examples are depicted (b). Spot number 23 displaying an experimental mass of 23 kDa was identified as agrin, a large glycoprotein (>200 kDa) of the extracellular matrix. Duplex Western blotting with an antiserum directed against the C-terminal fragment confirms the identity of spot 23 as an agrin fragment presumably generated by proteolytic processing (c).