DEVELOPMENT OF ONE AND TWO DIMENSIONAL ELECTROPHORESIS FOR THE ANALYSIS OF INDIVIDUAL PROTEIN TEAR SAMPLES

Vania Simonin

Abstract

PURPOSE. The determination of individual basal tear protein composition is important to monitor subtle changes taking place with contact lens wear and/or dry eye conditions. Several groups have achieved fine protein differentiation using electrophoresis, but required large tear volumes (>5 ul). The current paper reports on the optimisation of one and two dimensional electrophoresis separation techniques to achieve similar differential between proteins using low volume individual tear samples. METHOD. Basal tear protein (<1 ul) collected with glass capillaries were separated by one or two-dimensional electrophoresis (Phastsystem, Pharmacia) using non linear immobilized pH gradient gels pH 3-10 and SDS-PAGE 8-25% minigel with silver stain. The method was fully automated, producing results in less than 3 hours.

RESULTS. One dimension electrophoresis was carried both by molecular weight and by isoelectric points. These techniques enabled the separation of charge-based and size-based isoforms of the major proteins present on the tear film. The combination of these two separation techniques used in two dimensional electrophoresis lead to the identification of all the major proteins including tear lipocalins and albumin, various glycoproteins and some low MW (<15kDa) proteins. Such a technique could be used to i. Identify the role of lipocalins in stabilising the tear film in conjunction with tear lipids ii. Monitor the association between low grade inflammation and tear albumin. iii. Evaluate the assocation between lysozyme and lactoferrin levels and dry eye symptoms CONCLUSIONS. The technique developed and validated makes it possible to carry out a routine detailed analysis of tear protein composition on individual small volume tear samples (<1ul).

Details

Year: 2001

Program Number: Poster 125

Author Affiliation: Ecole Nationale Superieure de Chimie de Paris

Co-Authors: Cecile Maissa, Michel Guillon

Co-Author Affiliation: Optometric Technology Group, Optometric Technology Group

Room: Exhibit Hall C