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.
P81 cellulose papers
Manufactured by Merck Group
P81 cellulose papers are a type of laboratory filter paper designed for use in various chemical and biochemical applications. They are made of cellulose and are commonly used for separating and isolating compounds from complex mixtures. The papers provide a reliable and consistent filtration medium for a wide range of sample types.
Automatically generated - may contain errors
2 protocols using p81 cellulose papers
1
Purification and Acetylation Assay of Tip60
2
Purification and Acetylation Assay of Tip60
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!