Method Optimization for the Isolation of Total Protein From Human Conjunctival Epithelial Cells Collected via Impression Cytology

Abstract

Abstract: : Purpose: To optimize a technique for the isolation of total protein derived from human conjunctival epithelial cells collected in vivo via impression cytology. Methods: Conjunctival epithelial cells were collected via impression cytology from human volunteers using sterile Millipore (MP) membrane (pore size = 0.45µm) placed on the superior and temporal regions of the conjunctiva. Following collection, each MP membrane was frozen immediately at –70^oC. Total protein was isolated by laying the membrane on a glass plate, cell side up and cutting it into approximately 1 mm pieces. 10 ul of extraction buffer (50 mM Tris, 1% SDS, 30 mM dithiothreitol) was applied to the MP pieces and the pieces were placed in a 600 ul eppendorf tube. An additional 50 ul of extraction buffer was added to the tube which was then boiled for 10 minutes. The tube was spun at 14,000g for 6 minutes. The supernatant was collected into a separate eppendorf tube and frozen for future use. Total protein was quantified with BioRad’s RC DC protein assay. Western blotting of lipoxygenase type 2 protein (LOX2) was performed to confirm the collection of high quality total protein. Results: The average yield of protein from a single membrane ranged from 3 ug to 64 ug with a greater quantity of protein being collected from the superior conjunctival area of the eye. Positive identification of LOX2 protein from Western blot results confirms the collection of intact, high quality total protein. Conclusions: We conclude that the use of impression cytology using MP membranes followed by immediate freezing and then extraction and processing with methods optimized by our laboratory facilitates the collection of high quality total protein from human conjunctival cells. This method should prove very useful to assess the expression of a variety of proteins involved in both normal and pathophysiological functions of the human ocular surface.

Keywords: conjunctiva