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. 2009 Nov 20;4(11):e7932. doi: 10.1371/journal.pone.0007932

Figure 2. Validation of antibody specificity.

Figure 2

Sequence alignment (Smith-Waterman) of human CRX immunogen (amino acids 166 to 285) with OTX1 and OTX2 (A) demonstrates low identity for the relevant epitope (<40%). Residue number 1 correpsonds with residue 166 of CRX. AMAS conservation scores are listed below the alignment. Immunoblot of native, non-denatured GST-Crx, GST alone and with the unrelated protein GST-Cry1 demonstrates robust specificity of the H120 antibody for native Crx (B). Immunohistochemical analysis using the H120 anti-Crx antibody demonstrates strong reactivity in the developing retina of E14.5 mice, which are heterozygous for Crx (Crx +/−), while the signal is absent in the retina of Crx knockout mice (Crx −/−) (C). Competition assays with CRX protein on tissue sections of human retina and retinoblastoma (cells at top of image) demonstrate the immunoreactivity of the H120 anti-Crx antibody is specifically competed away with 1∶1 molar amounts of purified GST-Crx protein in normal and tumor cells.