Recombinant Human PCPE-1 Protein, CF Summary
Met1-Asp449, 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 MES and NaCl.|
|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.
- Dilution Buffer: 50 mM Tris, 5 mM CaCl2, pH 7.5
- Assay Buffer: 50 mM Tris, 5 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Recombinant Human PCPE-1 (rhPCPE-1) (Catalog # 2627-PE)
- Recombinant Human Pro Collagen I alpha 1 (rhCOL1A1) (Catalog # 6220-CL)
- Recombinant Human BMP‑1/PCP (rhBMP-1) (Catalog # 1927-ZN)
- Reducing SDS-PAGE Sample Buffer (3X or greater)
- SDS-PAGE or Western Blot
- Dilute rhCOL1A1 to 75 µg/mL in Dilution Buffer.
- Dilute rhPCPE-1 to 15 µg/mL in Assay Buffer.
- In a tube, combine equal volumes (a minimum of 15 µL of each is required for this assay) of diluted rhCOL1A1 and diluted rhPCPE‑1. Prepare samples in duplicate (one replicate to be used as a control).
- Incubate vials at 37 °C for 30 minutes.
- Dilute rhBMP-1 to 1.875 µg/mL in Assay Buffer.
- Add one equivalent (1/2 the total volume of rhCOL1A1/rhPCPE-1 mixtures) of diluted rhBMP-1 to each reaction tube. Prevent the reaction from occurring in the control duplicates by immediately mixing with reducing gel buffer (see step 8 for volumes).
- Incubate reaction tubes at 37 °C for 30 minutes.
- After incubation, combine 40 µL of each reaction mixture and control with 20 µL of reducing SDS-PAGE gel buffer. Mix and incubate samples at 95-100 °C for 3-5 minutes.
- Load 30 µL per lane and analyze the cleavage by SDS-PAGE followed by protein staining and/or Western blot.
- Activity calculation:
|% Cleavage = [1 -||% full-length rhCOL1A1 (reaction)||] x 100%|
|% full-length rhCOL1A1 (control)|
- rhPCPE-1: 0.1 µg
- rhCOL1A1: 0.5 µg
- rhBMP-1: 0.0125 µg
Procollagen C‑endopeptidase enhancers, known as PCPEs or PCOLCEs, are secreted extracellular matrix glycoproteins that consist of two CUB domains and one NTR domain. They are known to stimulate enzymatic cleavage of procollagens I‑III by the BMP‑1/tolloid family of metalloproteases, also known as procollagen C‑proteinases (1). PCPE‑1 is expressed primarily by interstitial connective tissues such as tendons, calvaria, and skin (2). Although BMP‑1/tolloid proteinases are involved in processing of multiple extracellular proteins, the enhancer activity of PCPE‑1 is specific to procollagens since it has no effect on BMP‑1/tolloid cleavage of other substrates (3). It is thought that PCPE‑1 enhances cleavage of procollagens by binding to the substrate and inducing a conformation change in the substrate (3), although interaction between PCPE‑1 and full‑length BMP‑1/tolloid proteinases has also been reported (4).
- Steiglitz, B.M. et al. (2002) J. Biol. Chem. 277:49820.
- Kessler, E. et al. (1990) Biochem. Biophys. Res. Commun. 173:81.
- Moali, C. et al. (2005) J. Biol. Chem. 280:24188.
- Ge, G. et al. (2006) J. Biol. Chem. 281:10786.
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