Recombinant Human Pro-Collagen II Protein, CF Summary
Gln26-Arg266 & Gly957-Leu1487, with an N-terminal 5-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 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 Pro-Collagen II (rhCOL-2A1) (Catalog # 7589-CL)
- Recombinant Human BMP‑1/PCP (rhBMP-1) (Catalog # 1927-ZN)
- Reducing SDS-PAGE Sample Buffer
- SDS-PAGE or Western Blot
- Dilute rhCOL-2A1 to 75 μg/mL in Dilution Buffer.
- Dilute rhBMP-1 to 10 μg/mL in Assay Buffer.
- Combine one volume of diluted rhCOL-2A1 with three volumes of diluted rhBMP-1. For controls, combine one volume of rhCOL-2A1 with three volumes of Assay Buffer, as well as three volumes of rhBMP-1 with one volume of Assay Buffer.
- Incubate reaction vials and controls at 37 °C for one hour.
- After incubation, combine rhCOL-2A1/rhBMP-1 reaction mixtures and controls with reducing SDS-PAGE gel buffer at a 2:1 (reaction mixture:gel buffer) ratio (v/v). Mix and incubate samples at 95-100 °C for 3-5 minutes to stop reactions.
- Load 40 μL (0.5 μg of rhCOL-2A1) per lane and analyze the cleavage by SDS-PAGE followed by protein staining and/or Western blot.
- Activity calculation:
% Cleavage = [1 -
|% full-length rhCOL-2A1 (reaction)||] x 100%|
|% full-length rhCOL-2A1 (control)|
- rhCOL-2A1: 0.5 µg
- rhBMP-1: 0.2 µg
Background: Collagen II
Type II collagen is an essential component of cartilage. It is essential for skeletal development and for the ability of cartilage to resist compressive forces. Type II collagen is a homotrimer of a1(II) chains. Defects in Type II collagen are the cause of numerous diseases, including chondrodysplasias and arthritis (1). This recombinant mini pro-a1(II) collagen consists of the N‑terminal propeptide, 108 Gly-X-Y repeats, and the C‑terminal propeptide. Proteolytic processing of the C‑terminal propeptide by the procollagen C-proteinase (BMP-1) releases chondrocalcin, a calcium binding protein.
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