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M. M. Jablonski, R. Gandrakota, F. Giorgianni, S. Beranova-Giorgianni, S. Nookala; In Search of the Identity of the XAP-1 Antigen. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5457.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the identity of the XAP-1 antigen. The XAP-1 antibody has been used my many investigators and is recognized as a marker of photoreceptor outer segment maturity, yet the antigen to which it binds remains unknown.
Previous studies documented that the XAP-1 antigen is a photoreceptor outer segment membrane-associated protein. To maximize our success of identifying this protein, we prepared outer segment enriched preparations from mouse retinas. Crude membrane and cytoplasmic fractions from the outer segment enriched preparation were generated using ultracentrifugation. Proteins were extracted using DDM and separated using SDS-PAGE. nanoLC-ESI-MS/MS analysis was performed on the bands from a silver-stained gel that corresponded to the immunoreative band from Western blots. Once a match was found for the protein, confirmatory Western blots and immunohistochemistry experiments were performed.
Western blots performed using the XAP-1 antibody indicated a single immunoreactive band at ~78kD in lystates from both total outer segment and crude membrane preparations. No immunoreactive band was present in the cytoplasmic lysate. Aliquots of the crude membrane fraction were run on multiple lanes of a single 7% gel, one lane of which was transferred to PVDF membrane and probed with the XAP-1 antibody. The remaining lanes were silver-stained using a protocol that is compatible with MS. A very careful alignment of the Western blot with the silver-stained lanes indicated the presence of a single lightly stained band at the same position as the immunopositive band. MS analysis of pooled silver stained bands determined that the protein in the band at 78kD is Grp78. Western blotting and immunohistochemistry both support the possibility that the XAP-1 antigen is Grp78. Additional studies are underway to confirm this possibility.
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