Purpose: In our initial attempt to identify differentiation markers for ocular surface epithelia, we observed a unique staining pattern by a commercial anti-Gαq antibody. We further isolate and characterize the protein reactive with this anti-Gαq antibody in human ocular surface epithelia. Methods: Human donor corneoscleral buttons were sectioned and stained with a battery of commercial antibodies against Gα proteins. Western blot analysis of cell lysates of corneal epithelial cells and HEK 293 cells transfected with Gαq cDNA was used to determine the identity of the protein reactive with the anti-Gαq antibody (E-17). Comparisons were made with another anti-Gαq antibody (G4415) and an anti-cytokeratin 12C (J7) antibody. The isolated proteins reactive with E17 and J7 were then analyzed with two dimensional isoelectric focusing. Polypeptide sequences were identified using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel protein digestion. Results: The E-17 anti-Gαq antibody preferentially stained the entire corneal epithelia and the suprabasal layers of the limbus with complete absence of staining in the basal limbus and conjunctiva. Western blot analysis of corneal epithelial cells showed that E-17 antibody identified a protein with a molecular weight of 55 kDa. However, the antibody did not react with the purported antigen, Gαq protein (42 kDa) produced by Gαq cDNA. Another anti-Gαq antibody (G4415) did not react with the 55 kDa protein but did react with the 42 kDa Gαq protein. Further comparison of the E-17 antibody with the J7 antibody revealed that both recognized the 55 kDa band in one and two dimensional analysis. MALDI-TOF MS analysis confirmed that the 55 kDa protein of interest was actually cytokeratin 12 (CK12), rather than Gαq protein. Conclusions: The commercial E-17 anti-Gαq antibody did not react with Gαq protein, its purported antigen. Instead, it recognized a 55 kDa protein, which was characterized to be cytokeratin 12 by isoelectric focusing and peptide fingerprinting with mass spectrometry. Based on its reactivity with CK12, this commercial E-17 can be used as a differentiation marker to study ocular surface epithelia.
|Number of pages||7|
|State||Published - Nov 16 2004|