Protein Extraction from Lens Capsules

Each lens capsule sample was sonicated for 30 sec (using the UP50H sonicator by Hielscher Ultrasonics GmbH, Teltow, Germany, connected with a 2-mm diameter MS2 microtip) in the presence of 92 μl double-distilled water (ddΗ₂Ο) and 8 μl 1% DOC stock solution (made in ddΗ₂Ο). Then, the sonicated homogenate was mixed with 300 μl ddΗ₂Ο, and the mixture was centrifuged at 16,000 ×g for 5 min at 4 °C to remove any insoluble material. The resulting supernatant was incubated for 10 min at 25 °C, followed by the addition of 46 μl ice-cold 100% TCA (final 10% TCA, 0.019% DOC), incubation for 15 min in an ice-water bath, and centrifugation at 16,000 ×g for 5 min at 4 °C. This DOC-TCA protein precipitation procedure is a modification of a procedure previously reported [87 (link)-89 (link)], which is able to precipitate minute quantities of proteins (as low as 3 µg) with ≥90% recovery. The resulting protein pellet was dissolved in 50 µl 50 mM NaOH containing 4 M urea and stored at −80 °C for measuring protein carbonyls and protein concentration.