Xenopus histone was acetylated with 3H-acetyl CoA (PerkinElmer) using a recombinant p300 as previously described (25 (link)). Unincorporated 3H-acetyl CoA was removed from p300-acetylated histones by dialysis using Slide-A-Lyzer Midi Dialysis Units (3500 MWCO, MilliporeSigma) before being used for histone deacetylation assay. Histone deacetylation assays were carried out at 30 °C for 60 min using 100 ng of WT (wildtype) or mutant HDAC3 proteins, 200 nM DAD, and 1 μM IP4 unless otherwise noted. For the gel-based assay, the product was detected by autoradiography following polyacrylamide gel electrophoresis (PAGE). For the liquid-based assay, ethyl acetate (MilliporeSigma) was used to extract the released 3H-acetic acid, which was quantified by liquid scintillation.
3h acetyl coa
Manufactured by PerkinElmer
3H-acetyl-CoA is a radiolabeled form of the essential coenzyme acetyl-CoA, which plays a central role in cellular metabolism. It is commonly used as a tracer in various biochemical and metabolic studies.
Automatically generated - may contain errors
Lab products found in correlation
Amplify solution, by GE Healthcare (4 mentions)
Tris hcl polyacrylamide gel, by Bio-Rad (4 mentions)
Cellulose papers, by Beckman Coulter (2 mentions)
Scintillation counter, by Beckman Coulter (2 mentions)
P81 cellulose papers, by Merck Group (2 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Acetyl coa, by PerkinElmer (1 mentions)
Flashplate, by PerkinElmer (1 mentions)
3h ac coa, by PerkinElmer (1 mentions)
Microbeta microplate counter, by PerkinElmer (1 mentions)
Tri carb 2910 tr, by PerkinElmer (1 mentions)
Sypro ruby, by Bio-Rad (1 mentions)
P81 phosphocellulose paper, by Cytiva (1 mentions)
Histone extraction kit, by Abcam (1 mentions)
Ls6500, by Beckman Coulter (1 mentions)
14 protocols using 3h acetyl coa
1
Histone Deacetylation Assay with Mutant HDAC3
2
Purification and Acetylation Assay of Tip60
3
Histone Acetylation Analysis Protocol
4
Histone Acetylation Assay of Nucleosomes
5
Nucleosome acetylation by p300
6
Quantitative Inhibition Assay for KAT8 Acetyltransferase
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KAT8 inhibition assays were performed using the previously purified
KAT8 (173–458, 5 nM), radio-isotope-labeled [3H]Acetyl-CoA
(Perkin Elmer, 3 μM) as the acetyl donor, and biotinylated H4
peptide (Sigma-Aldrich, 2.5 μM) as a substrate in a 100 mM NaCl,
40 mM Tris·HCl (pH 7.5), and 1 mM DTT assay buffer. Purified
KAT8 was incubated with the relevant inhibitor (1% DMSO final) in
a 2-fold serial dilution starting at 200 μM for 20 min at rt.
Then, [3H]Ac-CoA and biotinylated peptide were added to
a total assay volume of 50 μL and the reaction mixture was incubated
at rt for 4 h. The reaction was stopped, and the mixture was transferred
to a 96-well streptavidin scintillant coated FlashPlate (Perkin Elmer)
and incubated for 60 min at rt. The FlashPlate was washed with 0.1%
Tween-20, and the liquid scintillation counting (MicroBeta Microplate
Counter, Perkin Elmer) was employed to measure the radioactive signal.
Experiments were performed in biological triplicates. Signal reduction
compared to control was plotted (as inhibition percentage) as a function
of compound concentration and fitted by nonlinear regression (GraphPad
Prism 8.0) finally yielding the IC50 values.
KAT8 (173–458, 5 nM), radio-isotope-labeled [3H]Acetyl-CoA
(Perkin Elmer, 3 μM) as the acetyl donor, and biotinylated H4
peptide (Sigma-Aldrich, 2.5 μM) as a substrate in a 100 mM NaCl,
40 mM Tris·HCl (pH 7.5), and 1 mM DTT assay buffer. Purified
KAT8 was incubated with the relevant inhibitor (1% DMSO final) in
a 2-fold serial dilution starting at 200 μM for 20 min at rt.
Then, [3H]Ac-CoA and biotinylated peptide were added to
a total assay volume of 50 μL and the reaction mixture was incubated
at rt for 4 h. The reaction was stopped, and the mixture was transferred
to a 96-well streptavidin scintillant coated FlashPlate (Perkin Elmer)
and incubated for 60 min at rt. The FlashPlate was washed with 0.1%
Tween-20, and the liquid scintillation counting (MicroBeta Microplate
Counter, Perkin Elmer) was employed to measure the radioactive signal.
Experiments were performed in biological triplicates. Signal reduction
compared to control was plotted (as inhibition percentage) as a function
of compound concentration and fitted by nonlinear regression (GraphPad
Prism 8.0) finally yielding the IC50 values.
7
In vitro Acetylation Assay of NuA4 Complex
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In vitro acetylation assay was performed as described (54 (link)). Briefly, the acetylation was carried out in a 15 μl of reaction system containing 10 nM of the NuA4 complex, 100 nM of the GST-RPA complex (or 40 nM of GST-RFA1 or GST-RFA1-4KR), 0.25 μCi of 3H-acetyl CoA (Perkin Elmer), 10 mM Na Butyrate, 25 mM KCl, and 3 μl of 5× HAT buffer (250 mM Tris pH 8.0, 25% glycerol, 0.5 mM EDTA, 5 mM DTT, 5 mM PMSF). The reaction was incubated at 30°C for 30 min. The reaction product was spotted on a PVDF membrane for the liquid assay. After air drying, the membrane was washed three times with 50 mM carbonate buffer (0.5 M Na2CO3-NaHCO3, pH 9.2), followed by rinsing with acetone. The membrane was then placed in a scintillation vial, followed by the addition of a scintillation cocktail, and the radioactive signals were counted by a liquid scintillation analyzer (Tri-carb 2910TR, PerkinElmer).
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In vitro acetylation assay was performed as described (54 (link)). Briefly, the acetylation was carried out in a 15 μl of reaction system containing 10 nM of the NuA4 complex, 100 nM of the GST-RPA complex (or 40 nM of GST-RFA1 or GST-RFA1-4KR), 0.25 μCi of 3H-acetyl CoA (Perkin Elmer), 10 mM Na Butyrate, 25 mM KCl, and 3 μl of 5× HAT buffer (250 mM Tris pH 8.0, 25% glycerol, 0.5 mM EDTA, 5 mM DTT, 5 mM PMSF). The reaction was incubated at 30°C for 30 min. The reaction product was spotted on a PVDF membrane for the liquid assay. After air drying, the membrane was washed three times with 50 mM carbonate buffer (0.5 M Na2CO3-NaHCO3, pH 9.2), followed by rinsing with acetone. The membrane was then placed in a scintillation vial, followed by the addition of a scintillation cocktail, and the radioactive signals were counted by a liquid scintillation analyzer (Tri-carb 2910TR, PerkinElmer).
8
Nucleosome acetylation by p300
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Individual nucleosome variants (4.8 pmoles) were incubated with wild-type p300 (0.12 pmoles) in the presence of 5 μM 3H-acetyl-CoA (Perkin-Elmer) in 20 μL HAT buffer at 30 °C. The reactions were quenched after 0, 5, 15, and 45 min with 4x SDS sample buffer (final concentration: 50 mM Tris-HCl pH 6.8, 2 (w/v) % SDS, 10 (v/v) % glycerol, 1 (v/v) % 2-mercaptoethanol, and 12.5 mM EDTA, 0.02 (w/v) % bromophenol blue), and analyzed on a 15 % polyacrylamide Tris-HCl gel (Biorad) followed by protein staining using CBB and imaging. Additionally, a sample at the 45 min time-point was analyzed by native polyacrylamide gel electrophoresis (5 % acrylamide gel, 0.5 × TBE, 200 V, 40 min), followed by ethidium bromide staining and imaging. The gels were fixed (40 % EtOH, 10 % AcOH, 45 min, RT), incubated in Amplify solution (GE healthcare, 30 min, RT), and dried (2 h, 70 °C). 3H-acetyl incorporation was assessed by fluorography using a light-sensitive film. The experiment was performed in triplicate.
9
HAT Activity Assay for Rtt109, Asf1, and Histones
10
Purification and Histone Acetyltransferase Assay
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Full-length cDNA of Br140 was cloned into vector pBacPAK8 or pBacPAK8 carrying an N-terminal His-Flag tag. Full-length cDNA of elg1 was cloned into vector pBacPAK8 carrying an N-terminal double HA tag. Purification of the recombinant Enok and Br140 from Sf21 cells was performed as described previously (Huang et al. 2014 (link)).
Short oligonucleosomes were purified from HeLa cells as described previously (Utley et al. 1997 (link)). Reconstitution of recombinant nucleosomes and in vitro HAT assay were carried out essentially as described in the previous study (Huang et al. 2014 (link)). Briefly, nucleosomes were incubated with or without recombinant Enok and/or Br140 in HAT buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 100 µM EDTA, 10 mM sodium butyrate, 5% glycerol, 1 mM DTT, 100 µg/mL BSA, 1 mM acetyl-CoA, 1 mM PMSF) for 2 h at 27°C. For autoradiography (Fig. 1 C, bottom two panels), 0.4 µCi of 3H-acetyl-CoA (PerkinElmer) instead of 1 mM acetyl-CoA was used in 20-µL reactions, with a final concentration of 100 µM. The reactions were then adjusted using 2× SDS sample buffer to a final concentration of 1× (62.5 mM Tris-HCl at pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 143 mM β-mercaptoethanol) and heated for 6 min at 98°C followed by Western blotting or autoradiography.
Short oligonucleosomes were purified from HeLa cells as described previously (Utley et al. 1997 (link)). Reconstitution of recombinant nucleosomes and in vitro HAT assay were carried out essentially as described in the previous study (Huang et al. 2014 (link)). Briefly, nucleosomes were incubated with or without recombinant Enok and/or Br140 in HAT buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 100 µM EDTA, 10 mM sodium butyrate, 5% glycerol, 1 mM DTT, 100 µg/mL BSA, 1 mM acetyl-CoA, 1 mM PMSF) for 2 h at 27°C. For autoradiography (
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