Quantification of tear proteins by SDS-PAGE with an internal standard protein: A new method with special reference to small volume tears

Abstract

Quantitative determination of tear proteins is critical to our understanding of those ocular diseases with tear protein changes, but remains technically complex due to the small sample volumes available from patients. The aim of this study was to efficiently quantify the tear proteins by SDS-PAGE with an internal standard protein in small tear volumes. Schirmer test paper and capillary tubes were used to collect tear samples. Soybean trypsin inhibitor (SBTI) was used as an external standard or an internal standard to analyze tear samples in 15% SDS-PAGE gel. The total tear protein and its major components were quantified by band densitometry. Total tear protein concentrations were also measured by Bradford assay. Using this internal standard method, we compared differences between tear samples collected by the Schirmer test paper and capillary tube, and then examined the differences detected between tear samples obtained from young and elderly people. Using SBTI as an internal standard in SDS-PAGE, the total tear protein concentrations were determined to be 12.03 +/- 0.45 mg/ml, showing no difference from the result determined by the Bradford assay (P > 0.05). The quantities of the eight major tear protein bands were 2.26 +/- 0.07(18.8%), 0.10 +/- 0.01(0.8%), 0.63 +/- 0.13(5.3%), 0.52 +/- 0.07(4.3%), 1.14 +/- 0.18(9.5%), 1.33 +/- 0.21(11.1%), 2.76 +/- 0.16(23.0%), and 2.95 +/- 0.13 mg/ml (24.5%), similar to the result obtained by using SBTI as an external standard (P > 0.05). Comparing the methods of collection, we identified that the Schirmer test paper sampling induced increased concentrations of band 2 (mainly HSA) in tears, and decreased five other major tear protein bands. Comparing total protein concentrations among different age groups, we noted a higher total protein concentration in young people (P < 0.05). This high protein level was contributed equally by all major tear protein components. Using SBTI as an internal standard, we can simultaneously quantify total tear protein and the major tear protein components by SDS-PAGE densitometry using small volume tears. This method appears promising for use as a diagnostic tool for identifying the occurrence of ocular diseases with tear protein changes.