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Recombinant YTHDF1 protein

Expressed In

Baculovirus

Protein Species

Human

Cat No : 31608 31608

验证数据展示

  • Recombinant YTHDF1 protein gel. Recombinant YTHDF1 protein was run on an 8% SDS-PAGE gel and stained with Coomassie Blue. MW: 62.1 kDa Purity: > 80%

    Recombinant YTHDF1 protein gel. Recombinant YTHDF1 protein was run on an 8% SDS-PAGE gel and stained with Coomassie Blue. MW: 62.1 kDa Purity: > 80%

  • HTRF Assay for Recombinant YTHDF1 protein activity. 3 uM m6A ssDNA oligos (DNA sequence: 5’-GTTGG/ m6A/CTT-3’) were incubated with YTHDF1 protein in binding buffer including 50 mM HEPES-NaOH pH 7.0, 0.1% BSA for 1 hour at room temperature. Anti-FLAG antibody was used to detect reaction products.

    HTRF Assay for Recombinant YTHDF1 protein activity. 3 uM m6A ssDNA oligos (DNA sequence: 5’-GTTGG/ m6A/CTT-3’) were incubated with YTHDF1 protein in binding buffer including 50 mM HEPES-NaOH pH 7.0, 0.1% BSA for 1 hour at room temperature. Anti-FLAG antibody was used to detect reaction products.

  • Binding Assay for Recombinant YTHDF1 protein. 45 pmol biotinylated m6A ssDNA oligos (5’-GTTGCCTGTTCGTGTTGG/m6A/CTTGCCTGT-3’) were incubated with YTHDF1 protein in binding buffer for 3 hr at 4C. DNA-YTHDF1 compounds were incubated with streptavidin beads for 1 hour at 4C, and pull-down products were were run on a 10% SDS-PAGE gel, stained with Coomassie Blue. (Biotinylated,unmethylated ssDNA oligos used as negative control). YTHDF1 preferentially binds to ssDNA with m6A modifications compared with unmodified ssDNA.

    Binding Assay for Recombinant YTHDF1 protein. 45 pmol biotinylated m6A ssDNA oligos (5’-GTTGCCTGTTCGTGTTGG/m6A/CTTGCCTGT-3’) were incubated with YTHDF1 protein in binding buffer for 3 hr at 4C. DNA-YTHDF1 compounds were incubated with streptavidin beads for 1 hour at 4C, and pull-down products were were run on a 10% SDS-PAGE gel, stained with Coomassie Blue. (Biotinylated,unmethylated ssDNA oligos used as negative control). YTHDF1 preferentially binds to ssDNA with m6A modifications compared with unmodified ssDNA.

产品信息

Expressed In Baculovirus
Protein Species Human

Contents

Recombinant YTHDF1 protein is supplied at a concentration of 0.15 ug/ul in 25 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.04% Triton X-100, 0.2 mM TCEP.

背景介绍

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). YTHDF1 (YT521-B homology domain family 1, also known as C20orf21, DACA-1) is a member of the YTH (YT521-B homology) superfamily containing YTH domain. Human YTH domain family proteins include three members, YTHDF1-3, which mainly localized in the cytoplasm. YTHDF proteins comprise a highly conserved single-stranded RNA-binding domain which specifically recognizes and binds m6A-containing RNAs. A important role of YTHDF1 is regulating mRNA translation efficiency. YTHDF1 promotes ribosome loading to m6A-containing mRNAs and interacts with translation initiation factors eIF3 (EIF3A or EIF3B) to facilitate translation initiation.

Application Notes

Recombinant YTHDF1 protein is suitable for use in protein-protein interaction, RNA binding assay. HTRF Assay: 3 uM m6A ssDNA oligos (DNA sequence: 5’-GTTGG/ m6A/CTT-3’) were incubated with YTHDF1 protein in binding buffer including 50 mM HEPES-NaOH pH 7.0, 0.1% BSA for 1 hr. at room temperature. Anti-FLAG antibody was used to detect reaction products. Binding Assay: 45 pmol biotinylated m6A ssDNA oligos (5’-GTTGCCTGTTCGTGTTGG/m6A/CTTGCCTGT-3’) were incubated with YTHDF1 protein in binding buffer including 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 for 3 hr at 4C. The DNA-YTHDF1 compounds were incubated with streptavidin beads for 1 hour at 4C. YTHDF1 pulled down by streptavidin beads were run on a 10% SDS-PAGE gel and stained with Coomassie Blue. Biotinylated, unmethylated ssDNA oligos were used as negative control.. The binding assay result shows that YTHDF1 preferentially binds to ssDNA with m6A modi cation compared with unmodified ssDNA.

Protein Details

Recombinant YTHDF1 protein was expressed in a baculovirus expression system as the full length protein (accession number NP_060268.2) with an N-terminal FLAG-Tag. The molecular weight of YTHDF1 is 62.1 kDa.

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 guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.