In vitro methyltransferase assay was performed as described by Wu et al. (49 (link)). MLL2 complex was prepared by mixing equimolar (5 nM) concentrations of the core complex components, MLL2 (M342-381G-10; SignalChem), ASH2 (A372-30BG-50; SignalChem), WDR5 (W325-30H-50; SignalChem) and RBBP5 (R315-30H-50; SignalChem). Assays were performed in a volume of 20 μl comprising 4 μl of 5× HMTase buffer (250 mM Tris-HCl, pH 8.8, 2.5 mM DTT, 100 mM KCl, 50 mM MgCl2), 1 μl of protease inhibitor cocktail (Roche #11836170001), 4 μl of the MLL2 complex, 1 μl of s-adenosyl-L-methionine (SAM; 2 mM in water, MP Biomedicals, Inc.), 0.5 μl of 1 mg/ml recombinant Histone H3 (Millipore) and 100 ng of PASK full-length (TP-309096; Origene) or PASK kinase only active protein (PV3972; Thermofisher Scientific). For the assays with V5-purified PASK and mutants, PASK proteins were replaced with the respective mutant. For PASK inhibition by Bio E-115, the specified concentration of the inhibitor was pre-incubated at 37°C for 15 min with the assay components except for the substrate, SAM. After incubation at 37°C for 3 h, sodium dodecyl sulphate loading buffer was added to stop each reaction mixture. Methylation of H3 was determined by western blotting using specific antibodies.
Recombinant histone h3
Manufactured by Merck Group
Recombinant Histone H3 is a laboratory product produced by the Merck Group. It is a recombinant form of the histone H3 protein, a core component of the nucleosome in eukaryotic cells. The product is intended for use in research applications.
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Lab products found in correlation
2 protocols using recombinant histone h3
1
In vitro Methyltransferase Assay of MLL2 Complex
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In vitro methyltransferase assay was performed as described by Wu et al. (49 (link)). MLL2 complex was prepared by mixing equimolar (5 nM) concentrations of the core complex components, MLL2 (M342-381G-10; SignalChem), ASH2 (A372-30BG-50; SignalChem), WDR5 (W325-30H-50; SignalChem) and RBBP5 (R315-30H-50; SignalChem). Assays were performed in a volume of 20 μl comprising 4 μl of 5× HMTase buffer (250 mM Tris-HCl, pH 8.8, 2.5 mM DTT, 100 mM KCl, 50 mM MgCl2), 1 μl of protease inhibitor cocktail (Roche #11836170001), 4 μl of the MLL2 complex, 1 μl of s-adenosyl-L-methionine (SAM; 2 mM in water, MP Biomedicals, Inc.), 0.5 μl of 1 mg/ml recombinant Histone H3 (Millipore) and 100 ng of PASK full-length (TP-309096; Origene) or PASK kinase only active protein (PV3972; Thermofisher Scientific). For the assays with V5-purified PASK and mutants, PASK proteins were replaced with the respective mutant. For PASK inhibition by Bio E-115, the specified concentration of the inhibitor was pre-incubated at 37°C for 15 min with the assay components except for the substrate, SAM. After incubation at 37°C for 3 h, sodium dodecyl sulphate loading buffer was added to stop each reaction mixture. Methylation of H3 was determined by western blotting using specific antibodies.
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In vitro methyltransferase assay was performed as described by Wu et al. (49 (link)). MLL2 complex was prepared by mixing equimolar (5 nM) concentrations of the core complex components, MLL2 (M342-381G-10; SignalChem), ASH2 (A372-30BG-50; SignalChem), WDR5 (W325-30H-50; SignalChem) and RBBP5 (R315-30H-50; SignalChem). Assays were performed in a volume of 20 μl comprising 4 μl of 5× HMTase buffer (250 mM Tris-HCl, pH 8.8, 2.5 mM DTT, 100 mM KCl, 50 mM MgCl2), 1 μl of protease inhibitor cocktail (Roche #11836170001), 4 μl of the MLL2 complex, 1 μl of s-adenosyl-L-methionine (SAM; 2 mM in water, MP Biomedicals, Inc.), 0.5 μl of 1 mg/ml recombinant Histone H3 (Millipore) and 100 ng of PASK full-length (TP-309096; Origene) or PASK kinase only active protein (PV3972; Thermofisher Scientific). For the assays with V5-purified PASK and mutants, PASK proteins were replaced with the respective mutant. For PASK inhibition by Bio E-115, the specified concentration of the inhibitor was pre-incubated at 37°C for 15 min with the assay components except for the substrate, SAM. After incubation at 37°C for 3 h, sodium dodecyl sulphate loading buffer was added to stop each reaction mixture. Methylation of H3 was determined by western blotting using specific antibodies.
2
Histone Acetylation Assay Protocol
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HAT assays were performed in a 30 μL reaction medium, using either 0.5 μg of recombinant histone H3 (Millipore, Cat. #14-411), 2 μg of chicken core histones (Millipore, Cat. #14-411), 1 μg H3-H4 tetramer, 2 μg histone octamer or 2 μg mononucleosome, in the presence of 1 μCi of 3 H-acetyl-CoA (ARC, Cat. #0213A-50 µCi) or 200 µM acetyl-CoA. Enzymatic reactions were performed using 100 nM of purified FLAG-N121, and the same amount of FLAG-TEN, FLAG-TEN N338Y , FLAG-ΔN121 or commercial P300 protein. Reactions were incubated at 30°C for 2 h, and then 7.5 μL 5× SDS-PAGE sample buffer was added, followed by boiling for 5 min. After being resolved on 15% SDS-PAGE, aliquots were subjected to LC-MS/MS, autoradiography or immunoblotting. For autoradiography, proteins were separated and transferred to a PVDF membrane, using a semi-dry blotter (TE70, GE Life Sciences).
3 H signal was detected with a BioMax Transcreen Intensifying Screen LE (Sigma, Cat.
#Z374318) and BIOMAX MS films. For immunoblotting, the antibodies specific for different acetylated lysine residues were listed in Supplementary Table 9.
3 H signal was detected with a BioMax Transcreen Intensifying Screen LE (Sigma, Cat.
#Z374318) and BIOMAX MS films. For immunoblotting, the antibodies specific for different acetylated lysine residues were listed in Supplementary Table 9.
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