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D. R. Powell, M. Thangavelu, H. L. Chandler, K. K. Nichols, J. J. Nichols; Evaluation of Tear Film Protein Extraction Buffers. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5628.
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To determine if differences exist in the yields of chemically extracted tear film proteins as well as from proteins extracted from soft contact lenses.
Tear samples were taken from each eye of forty, non-dry eye, soft contact lens wearers using glass microcapillaries. The length of the tear volume was measured within each capillary and then the tear sample was ejected into 20µL buffer and frozen at -86°C. O2Optix (lotrafilcon B, Ciba Vision Inc.) contact lenses were then fitted to each subject and dispensed for at least 12 hours daily wear for 14 days followed by removal and storage at -86°C by the examiner. Capillary collected tear samples from each eye within a subject were randomly assigned to either no extraction or to one of four extraction buffers (prechilled acetone, 20% TCA, urea, or 50:50 0.2% TFA/ACN) in groups of ten. Contact lens samples were subjected to same extraction buffers as the capillary collected tear protein samples for each subject. Bradford assays were performed to determine protein concentration from capillary collected tear and contact lens samples. Nonparametric statistical tests were conducted to evaluate the outcomes.
For capillary collected tear samples, there were significant reductions in proteins observed when extracted with a buffer (all p ≤ 0.007). Except for acetone vs. urea (p = 0.290), there was a significant decrease in the amount of protein extracted from the contact lenses when comparing all extraction buffers (all p < 0.005).
Extracted tear protein from capillary collected tears provide significantly less available protein for further analyses than unextracted. TFA/ACN protein extraction from lotrafilcon B contact lenses offers the highest yield of available proteins compared to the other extraction methods employed in this study.
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