LEDGF and the H3KC36me3-modified nucleosome were mixed at a molar ratio of 2:1 and incubated for 1 hour on ice. The mixture was applied to a Superose 6 Increase 3.2/300 column equilibrated in gel filtration buffer (20 mM HEPES-Na pH 7.5, 50 mM NaCl, 1 mM DTT). The peak fraction was cross-linked with 0.05 % (v/v) glutaraldehyde on ice for 10 minutes and quenched for 10 min using 10 mM Tris-HCl (pH 7.5), 2 mM lysine and 8 mM aspartate. The sample was transferred to a Slide-A-Lyzer MINI Dialysis Unit 20,000 MWCO (Thermo Scientific), and dialyzed for 6 hours against 500 mL dialysis buffer (20 mM HEPES-Na pH 7.5, 50 mM NaCl, 1 mM DTT).
Slide a lyzer mini dialysis unit 20 000 mwco
Manufactured by Thermo Fisher Scientific
The Slide-A-Lyzer MINI Dialysis Unit 20,000 MWCO is a lab equipment product designed for dialysis of small-volume samples. It has a molecular weight cut-off of 20,000 MWCO, allowing the removal of small molecules and ions while retaining larger molecules.
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4 protocols using slide a lyzer mini dialysis unit 20 000 mwco
1
Structural Analysis of LEDGF-H3K36me3 Complex
2
Reconstituted Nucleosome Purification and Crosslinking
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Reconstituted nucleosome core particles and CHD4 were mixed at a molar ratio of 1:2. AMP-PNP was added at a final concentration of 1 mM and the sample was incubated for 10 min on ice. After 10 min compensation buffer was added to a final buffer concentration of 30 mM NaCl, 3 mM MgCl2, 20 mM Na⋅HEPES pH 7.5, 4% (v/v) glycerol, 1 mM DTT. The sample was applied to a Superose 6 Increase 3.2/300 column equilibrated in gel filtration buffer (30 mM NaCl, 3 mM MgCl2, 20 mM Na⋅HEPES pH 7.5, 5% (v/v) glycerol, 1 mM DTT). The elution was fractionated in 50 µL fractions and peak fractions were analyzed by SDS-PAGE. Relevant fractions containing nucleosome core particle and CHD4 were selected and cross-linked with 0.1% (v/v) glutaraldehyde. The crosslinking reaction was performed for 10 min on ice and subsequently quenched for 10 min using a final concentration of 2 mM lysine and 8 mM aspartate. The sample was transferred to a Slide-A-Lyzer MINI Dialysis Unit 20,000 MWCO (Thermo Scientific), and dialysed for 4 hr against 600 ml dialysis buffer (30 mM NaCl, 3 mM MgCl2, 20 mM Na⋅HEPES pH 7.4, 20 mM Tris⋅HCl pH 7.5, 1 mM DTT). The sample was subsequently concentrated using a Vivaspin 500 ultrafiltration centrifugal concentrator (Sartorius) to a final concentration of ~200–300 µM.
3
Structural Analysis of LEDGF-H3K36me3 Complex
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LEDGF and the H3KC36me3-modified nucleosome were mixed at a molar ratio of 2:1 and incubated for 1 hour on ice. The mixture was applied to a Superose 6 Increase 3.2/300 column equilibrated in gel filtration buffer (20 mM HEPES-Na pH 7.5, 50 mM NaCl, 1 mM DTT). The peak fraction was cross-linked with 0.05 % (v/v) glutaraldehyde on ice for 10 minutes and quenched for 10 min using 10 mM Tris-HCl (pH 7.5), 2 mM lysine and 8 mM aspartate. The sample was transferred to a Slide-A-Lyzer MINI Dialysis Unit 20,000 MWCO (Thermo Scientific), and dialyzed for 6 hours against 500 mL dialysis buffer (20 mM HEPES-Na pH 7.5, 50 mM NaCl, 1 mM DTT).
4
Reconstitution and Purification of Pol II-NCP Complex
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A RNA with the sequence /56-FAM/UCU CAC UGG A was purchased from Integrated DNA Technologies, resuspended in water, and stored at −80 °C. To reconstitute the Pol II–NCP complex, Pol II, extended NCP and RNA were mixed at a molar ratio of 1:0.5:0.66 and incubated for 10 min at 30 °C. Compensation buffer was added after 10 min to a final buffer of 150 mM KCl, 3 mM MgCl2, 20 mM Na·HEPES pH 7.5, 4% (v/v) glycerol, 1 mM DTT. For RNA extension, we added TFIIS (1:0.57, relative to Pol II) and NTPs (1 mM final concentration) and incubated for 30 min at 30 °C. The reaction was centrifuged (21,000g, 4 °C, 10 min), and applied to a Superose 6 Increase 3.2/300 column equilibrated in gel filtration buffer (100 mM NaCl, 3 mM MgCl2, 20 mM Na·HEPES pH 7.5, 4% (v/v) glycerol, 1 mM DTT). Peak fractions were analysed by SDS-PAGE and cross-linked with 0.1% (v/v) glutaraldehyde and incubated for 10 min on ice. The cross-linking reaction was quenched for 10 min using a concentration of 2 mM lysine and 8 mM aspartate. The sample was transferred to a Slide-A-Lyzer MINI Dialysis Unit 20,000 MWCO (Thermo Scientific), and dialysed for 6 h against 600 ml dialysis buffer (100 mM NaCl, 2 mM MgCl2, 20 mM Na·HEPES pH 7.4, 20 mM Tris·HCl pH 7.5, 1 mM DTT).
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