| Popis: |
Characterization of α-VSA sera. In order to specify the exact target sequence of the polyclonal antisera, the semi-conserved and the variable domain of the RIFIN variants RIF40 and RIF50 and of the STEVOR proteins PFL2610w and MAL13P1.7 were separately expressed as recombinant proteins. The RIFIN antisera α-RIF40.2, α-RIF44 and α-RIF50 (A) as well as the STEVOR α-PFL2610w, α-MAL13P1.7, α-PFC0025c and α-PFA0750w (B), which were generated by immunization with a protein spanning the semi-conserved and the variable domain, were subsequently used for Western blot analysis. Furthermore, the α-PfMC-2TM-SC serum generated solely against the semi-conserved region of PFF1525c was included in the analysis to check the specificity for its protein family (C). Approximately 20 ng of each recombinant protein or lysate from 1x107 cell membranes were loaded in each lane as indicated. All four antisera, α-RIF40.2, α-RIF50, α-PFL2610w and α-MAL13P1.7, are specific to the semi-conserved region of their own antigen. Furthermore, all antisera used in this study were shown to recognize the semi-conserved region of their own protein family, but are not cross-reactive with other VSA families. Unspecific cross-reactions of the small VSA antisera with the His-tag were excluded using an unrelated Entamoeba histolytica His-tagged protein recombinantly expressed under the same conditions (His-tag control) and lysate from uninfected red blood cells (RBC lysate). The recombinant proteins of all three small VSA families tend to form multimer complexes; accordingly bands corresponding in size to monomers are labelled with a single *, dimers with ** and trimers with *** as calculated by the size of the recombinant His-tagged proteins |
