GST-Tip60 was purified by glutathione-agarose beads using a standard protocol7 (link). Custom purification of His-ATF3 or His-ΔATF3 through a two-step protocol (IMAC affinity chromatography followed by SEC FPLC) was carried out by ProteinOne. The Tip60 HAT activity was assayed by incubating 0.5 μg of GST-Tip60 with 5 μl of [3H]-acetyl CoA (PerkinElmer) and 0.5 μg of recombinant histone H4 (Biolabs) with or without purified ATF3 in a buffer containing 50 mM Tri-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, and 10% glycerol for 1h at 30°C. Reaction mixtures were spotted onto P81 cellulose papers (Millipore) and the papers were washed for 6 times with 50 ml of 50 mM sodium phosphate (pH 9.0). After the final wash, the cellulose papers were air-dried and radioactivity was measured using a scintillation counter (Beckman). To measure Tip60 auto-acetylation, reaction mixtures were resolved by SDS-PAGE, and the [3H]-acetyl labeled Tip60 was visualized by autography.
Cellulose papers
Manufactured by Beckman Coulter
Cellulose papers are a type of laboratory equipment used for various applications in scientific research and analysis. They are made from cellulose, a natural polymer found in plant cell walls. These papers are designed to provide a surface for the separation, filtration, and analysis of samples. Cellulose papers are commonly used in chromatography, electrophoresis, and other analytical techniques.
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2 protocols using cellulose papers
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Purification and Acetylation Assay of Tip60
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Purification and Acetylation Assay of Tip60
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GST-Tip60 was purified by glutathione-agarose beads using a standard protocol7 (link). Custom purification of His-ATF3 or His-ΔATF3 through a two-step protocol (IMAC affinity chromatography followed by SEC FPLC) was carried out by ProteinOne. The Tip60 HAT activity was assayed by incubating 0.5 μg of GST-Tip60 with 5 μl of [3H]-acetyl CoA (PerkinElmer) and 0.5 μg of recombinant histone H4 (Biolabs) with or without purified ATF3 in a buffer containing 50 mM Tri-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, and 10% glycerol for 1h at 30°C. Reaction mixtures were spotted onto P81 cellulose papers (Millipore) and the papers were washed for 6 times with 50 ml of 50 mM sodium phosphate (pH 9.0). After the final wash, the cellulose papers were air-dried and radioactivity was measured using a scintillation counter (Beckman). To measure Tip60 auto-acetylation, reaction mixtures were resolved by SDS-PAGE, and the [3H]-acetyl labeled Tip60 was visualized by autography.
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