Recombinant Human CDC25B (aa 377-566) Protein, CF Summary
Product Specifications
Glu377-Gln566, 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.
1649-CD
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. 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: 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
- Dilute rhCDC-25B to 20 µg/mL in Assay Buffer.
- Dilute Substrate to 6 mM in Assay Buffer.
- 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.
- Incubate Reactions and Enzyme Control at 37 °C for 4 hours.
- Load 100 µL of Reactions into a plate and stop the reactions by adding 100 µL 0.2 M NaOH.
- Load 200 µL of Enzyme Controls into plate.
- Read plate at 410 nm (absorbance) in endpoint mode.
- 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
- rhCDC-25B: 1 µg
- pNPP: 1.5 mM
Reconstitution Calculator
Background: CDC25B
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.
- Draetta, G. and J. Eckstein (1997) Biochim. Biophys. Acta 1332:M53.
- Galaktionov, K. et al. (1995) Science 269:1575.
- Lammer, C. et al. (1998) J. Cell Sci. 111:2445.
- Miyata, H. et al. (2001) Cancer Res. 61:3188.
- Forrest, A. and B. Gabrielli (2001) Oncogene 20:4393.
- Gautier, J. et al. (1991) Cell 67:197.
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