Recombinant Human Pro-Collagen II Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Pro-Collagen II Protein, CF Summary

• Details of Functionality: Measured by the cleavage of its C-terminal propeptide by Recombinant Human BMP‑1/PCP (Catalog # 1927-ZN). >50% of full-length Pro-Collagen II is cleaved by Recombinant Human BMP‑1/PCP (Catalog # 1927-ZN), as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Collagen II protein
  Gln26-Arg266 & Gly957-Leu1487, with an N-terminal 5-His tag
• Accession #: P02458
• N-terminal Sequence: His & Asp1242
• Protein/Peptide Type: Recombinant Enzymes
• Gene: COL2A1
• Purity: >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 78 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 92-100 kDa, 65-70 kDa, 36 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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
  1. Dilute rhCOL-2A1 to 75 μg/mL in Dilution Buffer.
  2. Dilute rhBMP-1 to 10 μg/mL in Assay Buffer.
  3. 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.
  4. Incubate reaction vials and controls at 37 °C for one hour.
  5. 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.
  6. 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.
  7. Activity calculation:

  % Cleavage = [1 -	% full-length rhCOL-2A1 (reaction)	] x 100%
  	% full-length rhCOL-2A1 (control)

  Per Lane:
  • rhCOL-2A1: 0.5 µg
  • rhBMP-1: 0.2 µg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Pro-Collagen II Protein, CF

• Alpha-1 type II collagen
• ANFH
• AOM
• cartilage collagen
• Chondrocalcin
• COL11A3
• COL2A1
• Collagen 2
• collagen alpha-1(II) chain
• Collagen II
• collagen II, alpha-1 polypeptide
• collagen type 2
• collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphysealdysplasia, congenital)
• collagen, type II, alpha 1
• Collagen-2
• MGC131516
• SEDC

Background

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.

1. Kuivaniemi H. et al. (1997) Hum. Mutat. 9:300.

Publications for Collagen II (7589-CL)(1)

Publication using 7589-CL

• H Port, AC Bay-Jensen, Y He, MA Karsdal, T Gantzel, CS Thudium, S Holm Niels A Highly Sensitive Biomarker of Type II Collagen C-Terminal Pro-Peptide Associated with Cartilage Formation International Journal of Molecular Sciences, 2022-12-27;24(1):. 2022-12-27 [PMID: 36613894] (Cell Culture, Human)

Bioinformatics

• Gene Symbol: COL2A1
• Uniprot:
  • P02458(Human)