AlkB (RNA/DNA demethylase, Nuclease-free) (C007B-552296)

aladdin AlkB (RNA/DNA demethylase) is a high quality recombinant dioxygenase that is expressed and purified from E. coli. It effectively repairs various alkylation damages in RNA, ssDNA and dsDNA in an ɑ-ketoglutamate(ɑ-KG) and Fe(II) dependent manner. It is often used to remove methylation modifications, such as m6A, m1A and m3Cin RNA or DNA prior to qPCR analysisor high-throughput sequencing.s：Demethylation of m6A,m1A,m3Cand m1Gin RNA; Demethylation of m1A,m3C and m6A in DNA; Suitable for ARM-seq and PANDORA-seqetc.Unit definitionApplication：Applications:Demethylation of m6A,m1A,m3Cand m1Gin RNA; Demethylation of m1A,m3C and m6A in DNA; Suitable for ARM-seq and PANDORA-seqetc.Unit definition: One unit (U) is the amount of AlkB that removes 90-100% of the m6A modifications in total RNA of HEK 293T cells in 20 ul of reaction mixture at 37℃ after 100minutes of incubation. Under the regular conditions, the enzymatic activity of 1 μg of AlkB is approximately 1 unit.Source：Recombinant protein expressed in E. coli.Precautions：Keep AlkB on ice during the handling and return to -20℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation.Instructions for Use：1. Demethylation of m6A modificationsin total RNA by AlkB: a. Thaw and mix individual components before use and assemble reaction on ice as follows: Component Volume Final Concentration Nuclease-Free Water 9.2µl - Total RNA(0.05-4µg/µl) 4µl 10-800ng/µl AlkB Buffer Ⅰ 2µl 1× AlkB Buffer Ⅱ (10X) 2µl 1× RNaseInhibitor (40U/µl) 2µl 2U/µl AlkB(20µg/µl) 0.01-0.8µl 10-800ng/µl Total Volume 20µl - Note 1: Add same amount of RNA and AlkB in micrograms.After setting up the reaction mixture, mix well gently and spin down. When very small amount of AlkB is needed, AlkB dilution in Reaction Buffer (1×) can be used. Note 2:As RNA is easy to be degraded, all reagents and consumbles must be RNase free. Avoid any RNase contaminations during the operation.b. Incubation at 37℃ for 100min.c. Stop the reaction by adding an equal volume of stopping buffer containing 11mM EDTA and 200mM ammonium acetate to the reaction mixture, or by adding phenol chloroform. 2. Other uses can refer to the procedures mentioned above or other appropriate literatures.Appendix:N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional modification in eukaryotic RNA, and also exists in viral RNA. This modification is believed to account for more than 80% of RNA methylationspresent in mRNA, rRNA, tRNA, snRNA, microRNA (miRNA) and long non-coding RNA (lncRNA). The abundance of m6A in mRNA transcripts affects various metabolic processes of RNA ranging from splicing, nuclear export, translation ability and stability, and RNA transcription. The ɑ-KG and Fe(II) dependent AlkB catalyzes the conversion of m6A to adenine in vitro, through the formation of 6-hydroxymethyl derivative intermediate (HO-M6A).During the preparation of RNA-seq libraries, the modified nucleotides in RNA interfere with reverse transcription, resulting in transcription termination or transcription errors. Therefore, the methylated nucleotides present in RNA molecules can be demethylated with AlkB prior to reverse transcription, followed by regular steps of library preparation and high-throughput sequencing. This approach, known as AlkB-facilitated RNA methylation sequencing (ARM-Seq), can effectively improve the sensitivity and specificity of detecting methylatedRNAs and reveal more undiscovered small RNAs. AlkB, a bacteria reponsive protein to alkylating stresses, also repairs alkylation damages of DNA. It catalyzes the demethylation of 1-methyladenine (m1A) and 3-methylcytosine (m3C) in DNA in the presence of O2, Fe(II)and α-KG, during which α-KG is converted to succinate and the resulting unstable hydroxymethyl group is spontaneously released as formaldehyde.The N1 position of adenine and N3 position of cytosine in ssDNAare more susceptible to be methylated than those in double dsDNA. Therefore, AlkB has a strong preference for ssDNA. 600 chemiluminescent imaging system. As shown in the figure, AlkB efficiently removes m6A modifications in total RNA of HEK 293T cells. The m6A modifications in 4µg of HEK 293T cell total RNA are sufficiently removed by 4µg of AlkB after incubation at 37℃ for 100min.This photo is for reference only. The experiment result may vary due to different working conditions or other experimental factors.Related Products Cat. 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Specification: EnzymoPure™, Free from DNA endonuclease, exonuclease, RNase and phosphatase.