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With L-Cysteine; magnesium chloride In phosphate buffer at 37℃; for 0.25h; Enzymatic reaction;
2
[ 67-56-1 ]
[ 29908-03-0 ]
[ 1976-85-8 ]
[ CAS Unavailable ]
[ CAS Unavailable ]
[ CAS Unavailable ]
Yield
Reaction Conditions
Operation in experiment
6%
Stage #1: S-Adenosyl-L-methionine; uroporphyrinogen III With Tris-HCl buffer; Escherichia coli CR395 lysate; potassium chloride at 37℃; for 20h;
Stage #2: methanol With sulfuric acid for 18h;
Stage #1: S-Adenosyl-L-methionine; uroporphyrinogen III With Tris-HCl buffer; recombinant Pseudomonas denitrificans CobA; potassium chloride at 37℃; for 20h;
Stage #2: methanol With sulfuric acid for 18h;
With nuclear receptor binding SET [su(var) 3-9, enhancer of zeste, trithorax] domain-containing protein 1; magnesium chloride; Cleland's reagent In aq. buffer at 37℃; for 4h; Enzymatic reaction;
InVitro Peptide and Protein Methylation
General procedure: The membranes containing the peptides were washed for 20min in methylation buffer containing 50mM Tris-HCl (pH 9.0), 5mM MgCl2, and 4mM dithiothreitol (DTT) and incubated at room temperature for 60min in methylation buffer containing 50nM NSD1 and 0.76μM labeled [methyl-3H]-AdoMet (Perkin Elmer). Imaging and analysis was performed as described elsewhere (Kudithipudi etal., 2012; Rathert etal., 2008a, 2008b). Biotinylated peptide methylation reactions were performed as described elsewhere (Gowher etal., 2005). Protein methylation reactions were performed in methylation buffer with 0.76μM tritium-labeled AdoMet. Target proteins (2μM) were incubated with 100nM NSD1 for 4hr at 37°C. Reactions were stopped by boiling with SDS loading buffer at 95°C for 5min. Afterward, the samples were run on 16% or a specified percentage of SDS-PAGE, and the methylation was detected by autoradiography. For mass spectrometry experiments, the peptides were incubated in methylation buffer comprising 50mM Tris-HCl (pH 9.0), 5mM MgCl2, and 4mM DTT with 100nM NSD1 and 1mM AdoMet for 4hr at 37°C. The reactions were stopped by diluting the sample in 0.1% trifluoroacetic acid (TFA). The methylation of the peptides was assessed by mass spectrometry using a Bruker Autoflex Speed MALDI-TOF system as described later.