Sucrose gradient sedimentation analysis was performed as described39 (link). Briefly, 10–40% gradients were formed by layering 500 µl NEB1 buffer containing 10%, 20%, 30%, or 40% sucrose in a 11 × 34-mm centrifuge tube (Beckman) and allowed to equilibrate at room temperature for 2 h. Gradients were chilled, loaded with 500 µg MCF7 nuclear extract (adjusted to a volume of 150 µl) or 150 µl molecular weight markers (Sigma MW-GF-1000), and centrifuged in a Beckman TLS-55 rotor at 50,000 rpm (214,000×g) for 14 h. Thirty-six fractions of •45 µl were collected. For the markers, 20 µl of each fraction was electrophoresed and Coomassie stained. For the gradient fractions, 20 µl of fractions were analysed by immunoblotting using EZH2 (Cell Signaling Technology), SUZ12 (Abcam ab12073), TRIM37 (Abcam), RNF2 (Abcam ab101273) and BMI1 (Bethyl Laboratories, A301-694A).
Mw gf 1000
Manufactured by Beckman Coulter
The MW-GF-1000 is a laboratory instrument designed for molecular weight determination. It utilizes gel filtration chromatography to separate and analyze macromolecules, such as proteins and nucleic acids, based on their size and molecular weight.
Automatically generated - may contain errors
2 protocols using mw gf 1000
1
Nuclear Protein Complex Analysis via Sucrose Gradient
2
Nuclear Protein Complex Analysis via Sucrose Gradient
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Sucrose gradient sedimentation analysis was performed as described39 (link). Briefly, 10–40% gradients were formed by layering 500 µl NEB1 buffer containing 10%, 20%, 30%, or 40% sucrose in a 11 × 34-mm centrifuge tube (Beckman) and allowed to equilibrate at room temperature for 2 h. Gradients were chilled, loaded with 500 µg MCF7 nuclear extract (adjusted to a volume of 150 µl) or 150 µl molecular weight markers (Sigma MW-GF-1000), and centrifuged in a Beckman TLS-55 rotor at 50,000 rpm (214,000×g) for 14 h. Thirty-six fractions of •45 µl were collected. For the markers, 20 µl of each fraction was electrophoresed and Coomassie stained. For the gradient fractions, 20 µl of fractions were analysed by immunoblotting using EZH2 (Cell Signaling Technology), SUZ12 (Abcam ab12073), TRIM37 (Abcam), RNF2 (Abcam ab101273) and BMI1 (Bethyl Laboratories, A301-694A).
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