The assay was performed in 50 mM HEPES (pH 8.0), 50 mM NaCl, 1 mM EDTA, 5mM DTT and 0.2mM SAM. Each reaction was performed in 25 μL for 12 hours at 37°C with 300 ng of the corresponding peptide and 0.2 μL of active human recombinant PRMT5-MEP50 complex (Sigma, SRP0145) or water (negative control). The product of the reaction was desalted on a stageTip with 100 μL of 0.1% TFA and eluted in 30 μL of 50% ACN 0.1% FA. 2 μL of the desalted product was directly infused into LTQ Orbitrap XL using nanoAcquity UPLC (Waters) and analyzed at 100K resolution. The data was deconvoluted with Xtract with SN threshold 10, spectra extracted from the .xml outputs and plotted with the ggplot2 R package.
Prmt5 mep50 complex
Manufactured by Merck Group
The PRMT5-MEP50 complex is a protein complex consisting of the protein arginine methyltransferase 5 (PRMT5) and the methylosome protein 50 (MEP50). This complex is involved in the methylation of arginine residues on target proteins, which can regulate their function and localization.
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3 protocols using prmt5 mep50 complex
1
Methylation Assay of Recombinant PRMT5
2
Methylation Assay of Recombinant PRMT5
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The assay was performed in 50 mM HEPES (pH 8.0), 50 mM NaCl, 1 mM EDTA, 5mM DTT and 0.2mM SAM. Each reaction was performed in 25 μL for 12 hours at 37°C with 300 ng of the corresponding peptide and 0.2 μL of active human recombinant PRMT5-MEP50 complex (Sigma, SRP0145) or water (negative control). The product of the reaction was desalted on a stageTip with 100 μL of 0.1% TFA and eluted in 30 μL of 50% ACN 0.1% FA. 2 μL of the desalted product was directly infused into LTQ Orbitrap XL using nanoAcquity UPLC (Waters) and analyzed at 100K resolution. The data was deconvoluted with Xtract with SN threshold 10, spectra extracted from the .xml outputs and plotted with the ggplot2 R package.
3
Methylation Assay of ERα Domains
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The PRMT5/MEP50 complex (purchased from Sigma Aldrich) was incubated with the different domains of ERα fused to GST as already described (Le Romancer et al, 2008 (link)) in the presence of S‐adenosyl‐L [methyl‐3H] methionine ([3H] SAM 85 Ci/mmol from a 10.4 mM stock solution in dilute HCl/ethanol 9/1 [pH 2.0–2.5]; Perkin Elmer) for 90 min at 30°C. Methylation reactions were quenched by adding Laemmli sample buffer, heated at 95°C for 5 min, and separated by SDS–PAGE. Following electrophoresis, gels were soaked in Amplify reagent (Sigma) according to the manufacturer's instructions and visualized by autoradiography.
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