α crystallin

After optical evaluation, lenses were homogenised in 8 mL Tris-HCl buffer pH 8.4 containing 0.02% sodium azide water soluble as described in Section 2.5. Water soluble (WS) fractions were prepared as mentioned above in Section 2.5 and separated into fractions based on size using HPLC (Shimadzu LC-2010AHT, Shimadzu Corporation, Kyoto, Japan) comprising a quaternary pump, an automatic sampler, and a UV (280 nm) detector with data acquisition (Lab solutions software version 5.42 SP5, Shimadzu Corporation, Japan) and a size exclusion column, Yarra-SEC 4000 (3 μm; 300 mm × 7.8 mm, Phenomenex Corporation, UK), maintained at 40°C. The mobile phase comprised of 50 mM sodium phosphate pH 6.8/0.15 M sodium chloride. The isocratic flow rate was 1.0 mL/min. Purified α-crystallin was purchased from Sigma-Aldrich and used as a reference. The percentage (%) of soluble α-crystallin fraction was estimated as follows: $\begin{array}{c}\alpha \text{-crystallin\hspace{0.17em}\hspace{0.17em}fraction\hspace{0.17em}\hspace{0.17em}}\left(\%\right)=\frac{{\mathrm{A}\mathrm{U}\mathrm{C}}_{\mathrm{s}}}{{\mathrm{A}\mathrm{U}\mathrm{C}}_{\mathrm{c}}}\ast \mathrm{100},\end{array}$ where AUC_s and AUC_c are the area under the size exclusion (SE) chromatograms for α-crystallin fraction of the sample and negative control, respectively, and a representative chromatogram is shown as in Supplementary Figure 2 (in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3240261).