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. 2012 Sep 5;26(9):1976–1985. doi: 10.1038/leu.2012.125

Figure 2.

Figure 2

Selection of the PML and RARA constructs for raising anti-PML and anti-RARA antibodies. (a) To increase the chance of making an anti-PML antibody that worked in the immunobead assay, the PML immunogen had to meet specific criteria. The PML protein domains encoded by exon 1–3 (upstream of all known break points) were analyzed with various web-based programs to identify exposed regions (accessibility for the antibody), nonhomologous regions (no cross-reactivity with other human proteins) and antigenic determinants (difference between human and mouse). Accordingly, the first PML domain (first 46 amino acids) before the ring-type zinc finger domain was selected for cloning, protein expression, protein purification and subsequent immunization. The anti-PML antibody 5D8 was used in the immunobead assay. (b) Using the same selection criteria, the C-terminal domain of RARA (last 70 amino acids) were used for cloning, protein production and subsequent immunization. The anti-RARA antibody 9G4.16 was used in the immunobead assay.