Fig. 5.

Two-dimensional gel and immunoblot analyses of the pooled ion exchange chromatograph fractions (IEX groups 1 and 4) derived from Dermanyssus gallinae soluble mite extracts. (A–C) IEX group 1; (D–F) IEX group 4 proteins were separated on replicate gels with pI ranges of 7–11 (IEX group 1) and 3–10 (IEX group 4). Two replicate gels were electroblotted and screened with yolk-IgY from hens vaccinated three times with their respective IEX group 1 or 4 proteins (A and D, respectively) or with yolk-IgY from group 7 hens that had been immunised with adjuvant only (B and E). Bound IgY was detected with rabbit anti-IgY-peroxidase IgG and visualised with chemiluminescent substrate. A third gel replicate of IEX groups 1 and 4 (C and F) was stained with SimplyBlue™ SafeStain (Thermo Fisher Scientific) and the corresponding locations of the immuno-reactive spots and were identified and excised from the gel for liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) (circled and numbered).