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A Wohabrebbi, ES Umstot, A Iannaccone, DM Desiderio, MM Jablonski; Characterization of the XAP-1 antigen using differential centrifugation, second-dimensional gel electrophoresis, and MALDI-TOF MS . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1407.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To biochemically characterize the XAP-1 antigen, a unique photoreceptor protein. Methods:Eyes were removed from Xenopus laevis embryos and homogenized in HEPES buffer. The homogenate was subjected to differential centrifugation. Subcellular fractionations were collected and electrophoretically separated on a 1D gel for XAP-1 immunoblot analysis. Additional retinal samples were isoelectrically focused followed by 2D gel electrophoresis and Western blot analyses. Tryptic digestion of 2D gel XAP-1 antigen spots, in conjunction with MALDI-TOF MS, were performed to generate peptide-mass fingerprinting data of the XAP-1 antigen. XAP-1 antigen tryptic peptide masses were compared with those of known proteins in the SwissPROT and TrEMBL databases. Results:Western blot analysis detected the XAP-1 antigen in the soluble protein fraction. We identified five XAP-1 antigen spots on our 2D gel, at a relative molecular mass of 22KDa (monomer mass) with different iso-electric points, ranging between 7.3 and 8.9. A SwissPROT and TrEMBL database search using individual spot tryptic peptide masses demonstrated significant but varying degrees of homology with that of the lens structural proteins, the gamma-crystallins; a maximum of 73% sequence coverage of gamma-crystallin V was achieved with the tryptic peptide masses of spot 5, while values as low as 14% sequence coverage was obtained for homology between spot 1 and the same crystallin subtype. Conclusion:We identified the XAP-1 antigen as a soluble photoreceptor protein. While comparison of our tryptic peptide-mass fingerprint data with that of known proteins generated in silico suggests that the XAP-1 antigen is homologous to gamma-crystallins, our previous immunocytochemical data demonstrate distinct localization pattern in the eye. Further experiments are underway to determine the amino acid sequence of the XAP-1 antigen.
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