Recombinant Human METTL11A Protein, CF
Recombinant Human METTL11A Protein, CF Summary
Product Specifications
Thr2-Arg223, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
7159-MT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 25 mM HEPES, 2 mM MgCl2, 1 mM EDTA, pH 8.0
- Recombinant Human alpha -N-terminal Methyltransferase 1A/METTL11A (rhMETTL11A) (Catalog # 7159-MT)
- Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY (Catalog # 6466-AH)
- Glutathione, reduced (Amresco, Catalog # 399)
- Substrate: PPKQQLSKY (SM Biochemicals, Catalog # CUS326), 1 mM stock in deionized water
- Recombinant human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
- S-Adenosylmethionine (AdoMet) (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO
- ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates Inc., Catalog # T-003)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Prepare a 10 mM stock of reduced glutathione in deionized water.
- Prepare the standard curve by diluting the 10 mM reduced glutathione (10,000 pmol/µL) to 20 pmol/µL in Assay Buffer and performing six additional ½ serial dilutions.
- Dilute rhMETTL11A to 66.67 ng/µL in Assay Buffer.
- Dilute Substrate and S-Adenosylmethionine in Assay Buffer to 133.34 μM and 666.67 uM respectively.
- Combine equal volumes of dilute Substrate and S-Adensoylmethionine for the substrate mixture. Further dilute an appropriate amount of 133.34 μM Substrate to 66.67 μM in Assay Buffer for controls.
- Dilute rhAHCY and rhADA in Assay Buffer to 50 ug/mL and 88.9 μg/mL respectively.
- Combine equal volumes of dilute rhAHCY and rhADA for the rhAHCY/rhADA mixture.
- Load the microplate as follows:
- Reactions: 15 µL of 66.67 ng/µL rhMETTL11A, 15 µL of substrate mixture, and 20 µL of rhAHCY/rhADA mixture.
- Controls: 15 µL of 66.67 ng/µL rhMETTL11A, 15 µL of 66.67 uM Substrate, and 20 µL of rhAHCY/rhADA mixture
- Standard Curve: 50 µL per well
- Seal microplate and incubate at room temperature for 30 minutes.
- Dilute ThioGlo® to 100 µM in DMSO.
- After incubation, add 50 µL of 100 µM ThioGlo® to all wells used.
- Incubate at room temperature for 5 minutes in the dark.
- Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted thiol produced* (pmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the reduced glutathione standard curve using linear fitting and adjusted for Substrate Blank.
- rhMETTL11A: 1 μg
- rhAHCY: 0.5 µg
- Substrate: 10 µM
- S-Adenosylmethionine: 50 μM
- rhADA: 0.889 μg
- ThioGlo®: 50 µM
- Reduced Glutathione curve: 1000, 500, 250, 125, 62.5, 31.25 and 15.625 pmol
Reconstitution Calculator
Background: alpha-N-terminal Methyltransferase 1A/METTL11A
METTL11A, also named N‑terminal RCC1 methyltransferase (NRMT), is an alpha‑N‑methyltransferase that methylates the N‑terminal amino group of target proteins containing the N‑terminal motif [Ala/Pro/Ser]-Pro-Lys when the initiator Met is cleaved (1). It is responsible for N‑terminal methylation of RCC1, KLHL31, MYL2, MYL3, RB1, RPL23A and SET. NRMT lacks a SET domain but possesses a Rossman‑like a/b fold. The residues Asn169, Asp178, Asp181, and Ser183 of NRMT are important for substrate binding (2). RCC1 (Ran guanine nucleotide‑exchange factor) is the first protein for which any biological function of alpha‑N‑methylation by NRMT has been identified (3, 4). The multi‑spindle phenotype associated with either NRMT knockdown or methylation‑defective RCC1 mutants demonstrated the importance of alpha‑N‑methylation of RCC1 for normal bipolar spindle formation and chromosome aggregation (2). NRMT is robustly overexpressed in gastrointestinal cancers.
- Webb, K. J. et al. (2010) Biochemistry 49:5225.
- Christine, S. E. et al. (2010) Nature 466:1125.
- Chen, T. et al. (2007) Nature Cell Biol. 9:596.
- Hao, Y. et al. (2008) J. Cell. Biol. 182:827.
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