Recombinant Human P4HB Protein, CF Summary
Asp18-Lys505, with a C-terminal 10-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 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.
- Buffer A: 100 mM NaH2PO4, 3 mM DTT, pH 7.0
- Buffer B: 100 mM NaH2PO4, 3 mM EDTA, pH 7.0
- Recombinant Human Protein Disulfide Isomerase/P4HB (rhP4HB) (Catalog # 4236-DI)
- Insulin (Sigma, Catalog # I-5500), 10 mg/mL solution in 50 mM Tris, pH 7.5 (solution will be cloudy)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute insulin to 1.5 mg/mL in Buffer B.
- Incubate diluted insulin for 10 minutes at room temperature (insulin will solubilize).
- Dilute rhP4HB to 45 µg/mL in Buffer A.
- Mix 100 µL of 45 µg/mL rhP4HB and 200 µL of 1.5 mg/mL insulin. Create a Substrate Blank with 100 µL Buffer A and 200 µL insulin.
- Incubate at room temperature for 20 minutes.
- Load in a clear 96-well plate 100 µL from each reaction vial in duplicate.
- Read at 650 nm (absorbance) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (Abs/cm/min/mg) =
|Adjusted Vmax* (Abs/min)**|
|path corr.(cm)*** x amount of enzyme (mg)|
*Adjusted for Substrate Blank
**Note: the output of many spectrophotometers in kinetic mode is in mOD.
***Using the pathlength correction of 0.32 cm
- rhP4HB: 0.0015 mg
- Insulin: 0.100 mg
Background: Protein Disulfide Isomerase/P4HB
Protein Disulfide Isomerase, also known as prolyl 4‑hydroxylase subunit beta (P4HB), protocollagen hydroxylase, cellular thyroid hormone binding protein p55 and glutathione-insulin transhydrogenase (1-3) is an abundant multifunctional enzyme that belongs to the Protein Disulfide Isomerase family. It contains two thioredoxin domains that catalyze the formation, breakage and rearrangement of disulfide bonds. When present as a tetramer consisting of two alpha subunits and two beta subunits, this enzyme functions as a hydroxylase catalyzing the hydroxylation of prolyl residues in preprocollagen. P4HB has various additional functions (4-7). It binds thyroid hormone. It acts as a chaperone that inhibits aggregation of misfolded proteins. It plays a role in both the influx and efflux of S-nitrosothiol-bound nitric oxide. It is also a subunit of the microsomal triglyceride transfer protein complex.
- Pihlajaniemi, T. et al. (1987) EMBO J. 6:643.
- Cheng, S.Y. et al. (1987) J. Biol. Chem. 262:11221.
- Morris, J. I. and Varandani, P. T. (1988) Biochim. Biophys. Acta 949:169.
- Obata, T. et al. (1988) J. Biol. Chem. 263:782.
- Gilbert, H. F. (1997) J. Biol. Chem. 272:29399.
- Sliskovic, I. et al. (2005) J. Biol. Chem. 280:8733.
- Wetterau, J. R. et al. (1990) J. Biol. Chem. 265:9800.
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