Recombinant METTL3 / METTL14 complex

Catalog No: 31570 Format: 20 µg ¥88,000 Buy
Catalog No: 31970 Format: 1 mg ¥880,000 Buy
Expressed In: Baculovirus Protein Species: Human

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Contents

Recombinant METTL3 / METTL14 complex is supplied at a concentration of 0.45 µg/µl in 25 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.04% Triton X-100, 0.2 mM TCEP.

Background

N6-methylated adenine (m6A) is prevalently present in nearly all RNA types and can be found in all organisms from bacteria to humans. It preferentially appears around stop codons and within long internal exons in mammalian messager RNAs. m6A plays an important role in the efficiency of mRNA splicing, processing, translation efficiency, editing and mRNA stability. m6A also takes place in other RNA molecules, such as primary miRNA (pri-miRNAs).
In mammalian cells, METTL3 (methyltransferase-like 3, also known as IME4, M6A, MT-A70) and METTL14 (methyltransferase-like 14) are two m6A methyltransferases reported to date, which both contain methyltransferase domains. These two proteins form a stable heterodimer core complex of METTL3–METTL14 that functions in cellular m6A deposition on mammalian nuclear RNAs. METTL3 is the catalytically active subunit, while METTL14 plays a structural role critical for substrate recognition.

Application Notes

Recombinant METTL3 / METTL14 complex is suitable for use in enzyme kinetics, inhibitor screening, and selectivity profiling.

Protein Details

Recombinant METTL3 / METTL14 complex that includes full length human METTL3 (accession number NP_062826.2) without tag and full length METTL14 protein (accession number NP_066012.1) with an N-terminal FLAG-Tag was expressed in Sf9 cells. The molecular weights of METTL3 and METTL14 are 64.5 kDa and 53.3 kDa, respectively.

 

Recombinant METTL3 / METTL14 complex, protein gel..
Recombinant METTL3 / METTL14 complex was run on a 10% SDS-PAGE gel and stained with Coomassie Blue.
MW: METTL3: 64.5 kDa,
MW: METTL14: 53.3 kDa.
Purity: > 95%

MTase-Glo assay for METTL3 / METTL14 Complex m6A methyltransferase activity
1 µM Substrate RNA (5’-GUUGCCUGUUCGUGUUGGACUUGCCUGU-3’) and 1 µM SAM was incubated with different concentrations of METTL3 / METTL14 Complex in an 8 µl reaction system containing 50 mM Tris-HCl pH 8.6,0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP at room temperature for 1 hour (0.2 U/µL RRI was added in this system). 5×MTase-Glo Reagent was added to the products and incubated for 30 min. Then MTase-Glo Detectionwas added and luminescence were read after another 30 min incubation.SAH standard curve (0-2 µM) was performed following the same protocol.

MTase-Glo assay for METTL3 / METTL14 Complex m6A methyltransferase activity
1 µM Substrate RNA (5’-GUUGCCUGUUCGUGUUGGACUUGCCUGU-3’) and 1 µM SAM was incubated with different concentrations of METTL3 / METTL14 Complex in an 8 µl reaction system containing 50 mM Tris-HCl pH 8.6,0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP at room temperature for 1 hour (0.2 U/µL RRI was added in this system). 5×MTase-Glo Reagent was added to the products and incubated for 30 min. Then MTase-Glo Detectionwas added and luminescence were read after another 30 min incubation.SAH standard curve (0-2 µM) was performed following the same protocol.

Storage

Recombinant proteins in solution are temperature sensitive and must be stored at -80°C to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

Guarantee

This product is for research use only and is not for use in diagnostic procedures. This product is guaranteed for 6 months from date of arrival.