Recombinant Human CDC25B (aa 377-566) Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human CDC25B (aa 377-566) Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a substrate, p-Nitrophenyl phosphate (pNPP). The specific activity is >70 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human CDC25B protein
  Glu377-Gln566, with an N-terminal Met and 6-His tag
• Accession #: NP_004349
• N-terminal Sequence: Met
• Structure / Form: Based on mass spectrometric analysis, a carboxyl-terminal truncated peptide with a molecular mass of approximately 22.1 kDa may also be present in the recombinant protein preparation.
• Protein/Peptide Type: Recombinant Proteins
• Gene: CDC25B
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 23 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 23-25 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 10 mM DTT, pH 7.5
  • Recombinant Human CDC25B aa 377‑566 (rhCDC-25B) (Catalog # 1649-CD)
  • Substrate: p-Nitrophenyl phosphate (pNPP), 10 mM stock in deionized water
  • Sodium Hydroxide (NaOH), 0.2 M stock in deionized water
  • Clear 96-well Plate (Catalog # DY990)
  • Plate Reader with absorbance read capability
  1. Dilute rhCDC-25B to 20 µg/mL in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. Prepare reaction mixtures by combining equal volumes of dilute rhCDC-25B and dilute Substrate in microtubes. Include an Enzyme Control by combining dilute rhCDC-25B with twice the volume of 0.2 M NaOH, mix briefly; then add a volume of dilute Substrate equal to the volume of dilute rhCDC-25B. The Enzyme Control will have 2x the volume of the reaction mixture.
  4. Incubate Reactions and Enzyme Control at 37 °C for 4 hours.
  5. Load 100 µL of Reactions into a plate and stop the reactions by adding 100 µL 0.2 M NaOH.
  6. Load 200 µL of Enzyme Controls into plate.
  7. Read plate at 410 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:
  
  

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)) / (Incubation time (min) x amount of enzyme (µg))

  
  *Adjusted for Enzyme Controls
  **Derived using calibration standard p-Nitrophenol
  Per Well:
  • rhCDC-25B: 1 µg
  • pNPP: 1.5 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human CDC25B (aa 377-566) Protein, CF

• CDC25B
• CDC25HU2
• cell division cycle 25 homolog B (S. cerevisiae)
• cell division cycle 25 homolog B (S. pombe)
• cell division cycle 25B
• Dual specificity phosphatase Cdc25B
• EC 3.1.3.48
• M-phase inducer phosphatase 2

Background

Cell Division Cycle 25B (Cdc25B) phosphatase removes inorganic phosphate groups covalently attached to tyrosine, serine and threonine residues in proteins (1). Breast cancer patients bearing tumors containing high levels of Cdc25B have been found to have a greater incidence of aggressive, high‑grade tumors than those with low Cdc25B levels (2). In cells, the levels of Cdc25B activity are highest during the G2/M transition of the cell cycle, where it is suspected to be involved in “checkpoint” control of cell cycle progression (3). Overexpression of Cdc25B reduces the G2/M cell cycle block caused by ionizing radiation (4). Although activated by phosphorylation, Ser323 phosphorylation causes the enzyme to bind the protein 14-3-3, preventing substrate access to the catalytic site (5). One of the major substrates of Cdc25B is Cdc2, a kinase that is activated by dephosphorylation (6). The recombinant protein is truncated to remove the N-terminal regulatory domains and is fully active.

1. Draetta, G. and J. Eckstein (1997) Biochim. Biophys. Acta 1332:M53.
2. Galaktionov, K. et al. (1995) Science 269:1575.
3. Lammer, C. et al. (1998) J. Cell Sci. 111:2445.
4. Miyata, H. et al. (2001) Cancer Res. 61:3188.
5. Forrest, A. and B. Gabrielli (2001) Oncogene 20:4393.
6. Gautier, J. et al. (1991) Cell 67:197.
   

Bioinformatics

• Gene Symbol: CDC25B
• Uniprot:
  • NP_004349()