mRNA Cap 2'-O-Methyltransferase

Datasheet COA

价格￥570.00 （ 1-2周）

货号 UA070092

规格

2KU10KU

数量

产品介绍

产品介绍评论(0)

产品规格

分子别名

mRNA Cap 2'-O-Methyltransferase
表达宿主

E.coli
分子量

40 kDa (Reducing)
纯度

＞95% by SDS-PAGE and HPLC
活性

50 U/μl
标签

His Tag
性状

Liquid
缓冲体系

20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% Glycerol (pH 8.0 @ 25°C)
储存条件

Store at -25 ~ -15℃ for 2 years
文献引用

[1] Decombe A , El Kazzi P , Decroly E .Interplay of RNA 2'-O-methylations with viral replication[J].Current Opinion in Virology, 2023.
[2] Pastore B , Hertz H , Price I ,et al. pre-piRNA trimming and 2'-O-methylation protect piRNAs from 3' tailing and degradation in C.elegans.[J].Cell reports, 2021, 36(9):109640.

背景介绍

The mRNA Cap 2´-O-methyltransferase is a recombinant protein derived from vaccinia virus, it can add a methyl group to the 2'-O at the 5' end of the RNA immediately adjacent to the first nucleotide of the cap structure. This enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped Cap 0 to Cap 1. Cap 1 structure can enhance the translation efficiency of mRNA and reduce the immunogenicity of mRNA structure itself, so it helps to improve the expression level of encoded protein after mRNA transfection.

产品组分

Storage Solution: 50 U/μl mRNA Cap 2'-O-Methyltransferase in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% Glycerol (pH 8.0 @ 25°C)
10* Capping Buffer: 500 mM Tris-HCl, 50mM KCl, 10 mM MgCl2, 10mM DTT (pH 8 @ 25°C)

操作步骤

1. Combine uncapped RNA and nuclease-free water in a final volume of 14.0 μl.
2. Heat at 65°C for 5 minutes.
3. Place tube on ice for 5 minutes.
4. Add the following components in the order specified:

Components	Volume
Denatured uncapped RNA (from above)	14.0 μl
10* Capping Buffer	2.0 μl
GTP (10 mM)	1.0 μl
SAM (4 mM, dilute 32 mM stock to 4 mM)	1.0 μl
Vaccinia Capping Enzyme (10 U/μl)	1.0 μl
mRNA Cap 2´-O-Methyltransferase (50 U/μl)	1.0 μl

Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.
5. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).
6. Proceed with purification of the RNA (if required) for downstream applications.

注意事项

1. The capping reaction efficiency is affected by the structure of the RNA 5' end, so it is recommended to open the advanced structure of the RNA 5' end by thermal denaturation (heating at 65°C for 5 min, placing on ice for 5 min). Denaturation conditions can be adjusted according to the structural complexity of the 5' end of RNA. If the 5' end has no advanced structure, this step can be omitted.
2. The capping reaction can generally be completed within 60 min. If the RNA 5' end structure is complex or the RNA length is short (≤ 200 nt), the reaction time can be extended to 120 min.
3. SAM is unstable at pH 7-8, 37°C and needs to be freshly configured before the reaction starts. To avoid SAM degradation, the working solution needs to be stored on ice.
4. RNase inhibitors can be added to the reaction system to prevent RNase contamination, and the recommended concentration is 1-2 U/μl.
5. The Cap1 capped RNA of this product can add a Poly(A) sequence at the 3' end through Poly(A) polymerase to form a complete mRNA, which can be used for subsequent transfection experiments or in vitro translation experiments.

酶活定义

One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt long capped RNA transcript in 1 hour at 37°C.

生物活性
- The experiment was designed to add a cap and 2'-O-methylation to the 550nt RNA obtained by in vitro transcription in a one-step process, purify it, perform enzymatic digestion, and finally analyze the Cap 1 cap addition rate using LC-MS. The test results are shown in the chart.
电泳
- 2μg (R: reducing condition, N: non-reducing condition).
体积排阻色谱(SEC-HPLC)
- 97.5%
反相高效液相色谱(RP-HPLC)
- 98.9%