PURPOSE: To determine the identity of the XAP-1 antigen. The XAP-1 antibody has been used by many investigators and is recognized as an index of photoreceptor outer segment maturity, yet its antigen remains unknown. METHODS: Previous studies documented that the XAP-1 antigen is a photoreceptor membrane-associated protein. To enrich for this protein, the authors prepared outer segment preparations from mouse retinas. Crude membrane and cytoplasmic fractions from this preparation were then generated using ultracentrifugation. Proteins were solubilized using n-dodecyl beta-D-maltoside and separated using SDS-PAGE. Aliquots of the crude membrane fraction were run on multiple lanes of a single gel, one lane of which was transferred to PVDF membrane and probed with the XAP-1 antibody. The remaining lanes were silver-stained. 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. nanoLC-ESI-MS/MS analysis was performed on the pooled protein bands. On determining the protein identity, confirmatory Western blot analysis and immunohistochemistry studies were performed. RESULTS: Western blot analysis performed using the XAP-1 antibody indicated a single immunoreactive band at approximately 74 kDa in lysates from both total outer segment and crude membrane preparations. No immunoreactive band was present in the cytoplasmic lysate. MS analysis of pooled silver stained bands determined that the XAP-1 antigen is Grp78. Western blot analysis and immunohistochemistry both support this identification. CONCLUSIONS: Present evidence indicates that the XAP-1 antigen is Grp78, a protein that has been previously documented in the interphotoreceptor matrix surrounding cones.
PURPOSE: To determine the identity of the XAP-1 antigen. The XAP-1 antibody has been used by many investigators and is recognized as an index of photoreceptor outer segment maturity, yet its antigen remains unknown. METHODS: Previous studies documented that the XAP-1 antigen is a photoreceptor membrane-associated protein. To enrich for this protein, the authors prepared outer segment preparations from mouse retinas. Crude membrane and cytoplasmic fractions from this preparation were then generated using ultracentrifugation. Proteins were solubilized using n-dodecyl beta-D-maltoside and separated using SDS-PAGE. Aliquots of the crude membrane fraction were run on multiple lanes of a single gel, one lane of which was transferred to PVDF membrane and probed with the XAP-1 antibody. The remaining lanes were silver-stained. 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. nanoLC-ESI-MS/MS analysis was performed on the pooled protein bands. On determining the protein identity, confirmatory Western blot analysis and immunohistochemistry studies were performed. RESULTS: Western blot analysis performed using the XAP-1 antibody indicated a single immunoreactive band at approximately 74 kDa in lysates from both total outer segment and crude membrane preparations. No immunoreactive band was present in the cytoplasmic lysate. MS analysis of pooled silver stained bands determined that the XAP-1 antigen is Grp78. Western blot analysis and immunohistochemistry both support this identification. CONCLUSIONS: Present evidence indicates that the XAP-1 antigen is Grp78, a protein that has been previously documented in the interphotoreceptor matrix surrounding cones.
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