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Expressed In: Baculovirus Protein Species: Human
Contents
A representative Technical Data Sheet (TDS) is provided here. Please refer to the lot-specific TDS you will receive with your order for the lot-specific buffer contents and protein concentration.
Background
N6-methylated adenine (m6A) is prevalent 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 appears in other RNA molecules, such as primary miRNA (pri-miRNAs).
ALKBH5 (AlkB Homolog 5, RNA Demethylase, also known as OFOXD1, ABH5) specifically demethylates m6A RNA. ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles via demethylation activity.
Application Notes
Recombinant ALKBH5 protein is suitable for use in enzyme kinetics, inhibitor screening, and selectivity profiling.
3 µM m6A ssDNA oligos (DNA sequence: 5’-GTTGCCTGTTCGTGTTGG/m6A/ CTTGCCTGT-3’) were incubated with ALKBH5 protein in reaction buffer including 50 mM HEPES pH 7.5, 100 µM 2OG, 100 µM Ascorbate, 50 µM (NH4)2Fe(SO4)2·6H2O and 1 mM TCEP for 2 hours at room temperature. Dot-Blot was used for activity detection.
Protein Details
Recombinant ALKBH5 protein was expressed in a baculovirus expression system as the full length protein (accession number NP_060228.3) with an N-terminal FLAG-Tag. The molecular weight of ALKBH5 is 45.5 kDa.
Recombinant ALKBH5 protein run on an 8% SDS-PAGE gel stained with Coomassie Blue.
MW: 45.5 kDa
Purity: > 85%
Recombinant ALKBH5 protein activity.
3 µM m6A ssDNA oligos (sequence: 5’-GTTGCCTGTTCGTGTTGG/m6A/CTTGCCTGT-3’) incubated with ALKBH5 protein in reaction buffer for 2 hr at RT. Reaction products spotted onto positively charged nylon membrane were detected with Anti-m6A antibody (#31495). ALKBH5 only was used as negative control.
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.
