Recombinant Human PRL-3/PTP4A3 Protein, CF Summary
Ala2-Met173, with N-terminal Met and 7-His tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT 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 Buffer: 50 mM HEPES, 10 mM DTT, pH 7.5
- Recombinant Human PRL-3/PTP4A3 (rhPTP4A3) (Catalog # 8455-PT)
- Substrate: p-Nitrophenyl phosphate (Sigma, Catalog # N2765), 10 mM stock in deionized water
- NaOH, 0.2 M in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhPTP4A3 to 40 µg/mL in Assay buffer.
- Dilute Substrate to 5 mM in Assay buffer.
- Prepare reaction mixtures by combining equivalent volumes of dilute rhPTP4A3 and dilute Substrate in microtubes. Include an Enzyme Control by combining dilute rhPTP4A3 with twice the volume of 0.2 M NaOH, mix briefly, then add a volume of dilute Substrate equivalent to the volume of rhPTP4A3. The Enzyme Control will have 2x the volume of the reaction mixture.
- Incubate Reactions and Enzyme Controls at 37 °C for 24 hours.
- Load 100 µL of Reactions into a plate in triplicate and stop the reactions by adding 100 µL 0.2 M NaOH.
- Load 200 µL of Enzyme Controls into plate in triplicate.
- 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 (Sigma, Catalog # 241326).
- rhPTP4A3: 2 µg
- pNPP: 1.25 mM
Phosphatase of regenerating liver 3 (PRL-3), also known as protein-tyrosine phosphatase 4A3 (PTP4A3), is a member of the PRL subgroup of PTPases (1). It is preferentially expressed in skeletal muscle and the heart (2). Human PRL-3 shares 97% amino acid sequence identity with mouse and rat PRL-3. Structurally, PRL-3 consists of a five-stranded beta -sheet and six alpha -helices (3, 4). It has been shown to associate with membranes in a farnesylation-dependent manner (5). Both PRL-3 over-expression and attenuation of PRL-3 expression results in cell cycle arrest, suggesting that basal expression levels of this enzyme are important for normal cell cycle progression (6). PRL-3 has been shown to activate NF-kappa B signaling and may itself be regulated by FKBP38 (7, 8). PRL-3 also promotes epithelial to mesenchymal transition, tumor angiogenesis, cell migration, invasion, and metastasis, and it is over-expressed in multiple human cancers (9-18). Src-mediated phosphorylation of PRL-3 may be required for PRL-3-dependent cell migration and invasion (19).
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- Fiordalisi, J.J. et al. (2013) PLoS One 8:e64309.
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