Protein lobind tube

Lipase was extracted from different batches of dry roots. Extracts were diluted 1/200 either in 50 mM glycine buffer at pH 2.2, 0.15 M NaCl or in 50 mM acetate buffer at pH 5, 0.15 M NaCl. The diluted extracts were kept in Protein LoBind^® Eppendorf tubes at room temperature and assayed for lipase activity after 1, 3, 6, 10, and 14 days. After 14 days, the solutions were transferred into new tubes and the lipase activity was assayed 24 h later. The activities in stored extracts were expressed as a percentage of activity related to freshly diluted extracts. To test for lipase binding to tubes, 100 µL of lipase diluted 1/200 in one or the other above buffer were placed in Protein LoBind^® Eppendorf tubes for 24 h. The solution was removed and the tubes were rinsed with 100 µL of buffer of the same composition as the one used to dilute the extracts. One hundred µL of MUO substrate solution was placed for one min in the empty tubes. One hundred µL of stop solution was added and the fluorescence of the MU (4-methylumbelliferone) was measured, indicating the activity of lipase bound to the tubes.

Cardiac tissue was first homogenized on ice using a Polytron homogenizer in 10 volumes (mL/g tissues) of native wash buffer (5 mM NaH₂PO₄, 5 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 125 mM L-Met (pH 7.5), 1 mM PMSF and 1X MS-Safe protease and phosphatase inhibitor cocktail. The homogenate was centrifuged at 17,000 × g for 3 min at 4°C, and the supernatant was discarded. The washing and homogenization step was repeated once more, and then supernatant was discarded. To extract proteins such as the cardiac troponin (I-T-C) complex from human heart tissues without potentially denaturing the proteins’ tertiary or quaternary structure, a high ionic strength LiCl buffer at physiological pH was used (25 mM Tris (pH 7.5), 700 mM LiCl, 125 L-Met (pH 7.5), 1 mM PMSF and 1X MS-Safe protease and phosphatase inhibitor cocktail. The resulting pellet was washed, centrifuged, and homogenized in 5 vol (mL/g tissue) of LiCl native extraction buffer, then incubated at 4 °C for 10 min to extract the sarcomeric proteome. The homogenate was centrifuged at 17,000 × g for 3 min at 4°C and the supernatant containing the sarcomeric proteome was transferred to new Eppendorf Protein Lo-Bind tubes, and further centrifuged at 21,000 × g for 30 mins at 4 °C to clarify the extracts. The supernatants were finally transferred to new Eppendorf Protein Lo-Bind tubes, snap-frozen in liquid nitrogen, and stored at −80°C.

Tear film samples were collected in the morning hours, and when the tear wetted 30 mm on the Schirmer strips, the same ones used for STT-1, the strips were immediately placed in a 0.5 mL microtube (Protein LoBind Tubes; Eppendorf, São Paulo, Brazil) and placed in a thermal box until centrifugation. Immediately before centrifugation, the bottom of each microtube was punctured, and it was inserted into a larger 2.0 mL microcentrifuge tube (Protein LoBind Tubes; Eppendorf, São Paulo, Brazil) for extracting the tear fluid, as described by [16 (link)]. The TF was obtained through centrifugation (25.830 g for 10 min at 4°C) of the Schirmer strips (Ophthalmos, São Paulo, Brazil) and placed in 1.5 mL microtubes.
As tear sample amount of each animal has low volume, it was necessary to mix the individual samples of the 12 cats, place in a single 1.5 mL microtube, and keep frozen at a temperature of −20°C, until protein preparation for two-dimensional polyacrylamide gel (2D-SDS-PAGE).

For the proteomic experiments, MCF-7 cells were cultured as described in the cell culture section before and transferred to T-25 cm² flasks. After one day of incubation, cells were treated with XN, XNC, and DMSO as control. For the treatment, the IC₅₀ concentration determined by antiproliferative assays was used, respectively (XN: 12.25 μM, XNC: 4.18 μM). After two more days of incubation, medium was removed completely and cells were washed two times with PBS. Finally, 1 mL PBS was added, cells were scraped from the surface of the flask and transferred to an Eppendorf Protein LoBind Tube (Eppendorf, Germany). Cells were centrifuged at 16000 rcf, for 10 min, and 4°C. Supernatant was completely discarded and the pellet was shock frozen in dry ice, containing 80% EtOH and stored at -80°C until protein extraction was performed. The pelletized MCF-7 cells were sampled in quadruplicates.