Transfected HEK293T cells were lysed in a Triton X-100-based buffer (see Western blot), and the lysates were loaded on a linear sucrose density gradient, which had formed via equilibration between a 50% (w/v) and a 12.5% sucrose solution in a 13 × 51 mm centrifuge tube (Beckman Coulter) upon horizontal incubation for 3 h at RT62 (link). After centrifugation (201,000g/16 h/25 °C) in an Optima MAX Ultracentrifuge using an MLS-50 rotor (Beckman Coulter), 20 fractions of equal volume were collected, and every second fraction was analyzed by Western blotting (see above).
51 mm centrifuge tube
Manufactured by Beckman Coulter
The 13 × 51 mm centrifuge tube is a laboratory equipment used for the separation of different components within a liquid sample through the process of centrifugation. It is a cylindrical container with a capacity of approximately 50 milliliters.
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2 protocols using 51 mm centrifuge tube
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Sucrose Density Gradient Fractionation
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Sucrose Density Gradient Fractionation
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For sucrose density gradient preparation, 2 ml of a 50% (w/v) sucrose solution was overlaid with 2 ml of a 12.5% (w/v) sucrose solution in a 13 × 51 mm centrifuge tube (Beckman Coulter), and placed horizontally for 3 h at RT to allow equilibration of a linear density gradient39 (link) before overlaying with the sample. Centrifugation was performed using a MLS-50 rotor in an Optima MAX Ultracentrifuge (Beckman Coulter) at 50,000 r.p.m. for 16 h at 25 °C. Afterwards, 20 fractions of equal volumes were collected from top to bottom, and indicated fractions were analyzed by western blotting (see above).
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