Recombinant Human Sonic Hedgehog/Shh (C24II) N-Term GMP, CF Summary
Cys24-Gly197 (Cys24Ile-Ile), with an N-terminal Met
Produced using non-animal reagents in an animal-free laboratory. Manufactured and tested under current Good Manufacturing Practice (GMP) guidelines.
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||Lyophilized from a 0.2 μm filtered solution in PBS and NaCl.|
|Reconstitution||Reconstitute at 100-200 μg/mL in PBS, and allow up to 24 hours at 2 to 8 °C for complete reconstitution.|
|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.
1 μg/lane of GMP-grade Recombinant Human Sonic Hedgehog/Shh (C24II) N-Term (Catalog # 1845-GMP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing R band at 21.5 kDa and NR band at 22.5 kDa, respectively.
GMP-grade Recombinant Human Sonic Hedgehog/Shh (C24II), N-Terminus (Catalog # 1845-GMP) induces alkaline phosphatase production by the C3H10T1/2 mouse embryonic fibroblast cell line. The ED50 for this effect is 0.1-0.4 μg/mL.
LC/ESI-MS analysis of GMP-grade Recombinant Human Sonic Hedgehog/Shh (C24II) N-Terminus (Catalog # 1845-GMP). The major peak at 19809 corresponds to the calculated molecular mass, 19814 Da.
Background: Sonic Hedgehog/Shh
Sonic Hedgehog (Shh) is expressed in embryonic tissues that are critical for the patterning of the developing central nervous system, somite, and limb. It is also involved in whisker, hair, foregut, tooth, and bone development. Shh regulates neural and hematopoietic stem cell fate and is important for thymocyte differentiation and proliferation as well as T cell determination. In adult tissue Shh is associated with cancer development and tissue remodeling following injury (1-3). Human Shh encodes a 462 amino acid (aa) precursor protein that is autocatalytically processed to yield a non-glycosylated 19 kDa N-terminal fragment (Shh-N) and a glycosylated 25 kDa C-terminal protein (Shh-C) (4). Shh-C, which is responsible for the intramolecular processing of Shh, is rapidly degraded following Shh proteolysis (5). Shh-N is highly conserved, sharing >98% aa identity between mouse, human, rat, canine, porcine, and chicken Shh-N. Shh-N can be palmitoylated at its
N-terminal cysteine and modified by cholesterol addition at its C-terminus (6). These modifications contribute to the membrane tethering of Shh as well as its assembly into various sized multimers (6-9). Lipid modification and multimerization greatly increase Shh-N receptor binding affinity and signaling potency (5, 6, 8, 9). Monomeric and multimeric Shh can be released from the plasma membrane by the cooperative action of DISP1, SCUBE2, and TACE/ADAM17 (10-12). Modifications also extend the effective range of Shh functionality and are required for the development of protein gradients important in tissue morphogenesis (9, 13). Canonical signaling of Shh is mediated by a multicomponent receptor complex that includes Patched (PTCH1, PTCH2) and Smoothened (SMO) (14). The binding of Shh to PTCH releases the basal repression of SMO by PTCH. Shh activity can also be regulated through interactions with heparin, glypicans, and membrane-associated Hip (hedgehog interacting protein) (13, 15, 16).
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- Chen, M.-H. et al. (2004) Genes Dev. 18:641.
- Etheridge, L.A. et al. (2010) Development 137:133.
- Jakobs, P. et al. (2014) J. Cell Sci. 127:1726.
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- Carpenter, D. et al. (1998) Proc. Natl. Acad. Sci. USA 95:13630.
- Filmus, J. and M. Capurro (2014) Matrix Biol. 35:248.
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Manufacturing SpecificationsGMP Proteins
R&D Systems, a Bio-Techne Brand's GMP proteins are produced according to relevant sections of the following documents: WHO TRS, No. 822, 1992 Annex 1, Good Manufacturing Practices for Biological Products; USP Chapter 1043, Ancillary Materials for Cell, Gene and Tissue-Engineered Products and USP Chapter 92, Growth Factors and Cytokines Used in Cell Therapy Manufacturing.
R&D Systems' quality focus includes:
- Manufactured and tested under an ISO 9001:2015 and ISO 13485:2016 certified quality system
- Documented processes and QA control of documentation and process changes
- Personnel training programs
- Raw material testing and vendor qualification/monitoring
- Fully validated equipment, processes and test methods
- Equipment calibration schedules using a computerized calibration program
- Facility maintenance, safety programs and pest control
- Material review process for variances
- Monitoring of stability over product shelf-life
R&D Systems strives to provide our customers with the analytical characteristics of each product so that customers may determine whether our products are appropriate for their research. The Certificate of Analysis provided contains the following lot specific information:
- N-terminal amino acid analysis, SDS-PAGE analysis, and endotoxin level (as determined by LAL assay) performed on each bulk QC lot, not on individual bottlings of each QC lot
- Post-bottling lot-specific bioassay results (compliance with an established range) and results of microbial testing according to USP
- Host Cell Protein testing performed by ELISA
- Mycoplasma testing by ribosomal RNA hybridization assay
Additional testing and documentation requested by the customer can be arranged at an additional cost.
Production records and facilities are available for examination by appropriate personnel on-site at R&D Systems in Minneapolis, Minnesota USA.
R&D Systems sells GMP grade products for preclinical or clinical ex vivo use. They are not for in vivo use. Please read the following End User Terms prior to using this product.
Animal-Free Manufacturing Conditions
Our dedicated controlled-access animal-free laboratories ensure that at no point in production are the products exposed to potential contamination by animal components or byproducts. Every stage of manufacturing is conducted in compliance with R&D Systems' stringent Standard Operating Procedures (SOPs). Production and purification procedures use equipment and media that are confirmed animal-free.
- All molecular biology procedures use animal-free media and dedicated labware.
- Dedicated fermentors are utilized in committed animal-free areas.
- Protein purification columns are animal-free.
- Bulk proteins are filtered using animal-free filters.
- Purified proteins are stored in animal-free containers in a dedicated cold storage room.
- Low Endotoxin Level.
- No impairment of biological activity.
- High quality product obtained under stringent conditions.
Product Specific Notices
The End User is aware that R&D Systems, Inc. sells GMP products for preclinical or clinical ex vivo use and not for in vivo use. The End User further agrees, as a condition of the sale of R&D Systems' GMP products that: a) the End User will not use this GMP Product in any procedure wherein the product may be directly or indirectly administered to humans, unless the End User has obtained, or prior to their use will have obtained, an Investigational New Drug (IND) exemption from the FDA and will use the product only in accordance with the protocols of such IND and of the Institutional Review Board overseeing the proposed research, or b) the End User will use the products outside of the United States in accordance with the protocols of research approved by the Institutional Review Board or authorized ethics committee and regulatory agencies to which the End User is subject to in their territory.
TERMS AND CONDITIONS
The following limitation applies to R&D Systems' warranty and liability for damages: All products are warranted to meet R&D Systems' published specifications when used under normal laboratory conditions.
R&D SYSTEMS DOES NOT MAKE ANY OTHER WARRANTY OR REPRESENTATION WHATSOEVER, WHETHER EXPRESS OR IMPLIED, WITH RESPECT TO ITS PRODUCTS. IN PARTICULAR, R&D SYSTEMS DOES NOT MAKE ANY WARRANTY OF SUITABILITY, NONINFRINGEMENT, MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE.
NOTWITHSTANDING ANY OTHER PROVISIONS OF THESE TERMS AND/OR ANY OTHER AGREEMENT BETWEEN R&D SYSTEMS AND PURCHASER FOR THE PURCHASE OF THE PRODUCTS, R&D SYSTEMS' TOTAL LIABILITY TO PURCHASER ARISING FROM OR IN RELATION TO THESE TERMS, AN AGREEMENT BETWEEN THE PARTIES OR THE PRODUCTS, WHETHER ARISING IN CONTRACT, TORT OR OTHERWISE SHALL BE LIMITED TO THE TOTAL AMOUNT PAID BY PURCHASER TO R&D SYSTEMS FOR THE APPLICABLE PRODUCTS. IN NO EVENT WILL R&D SYSTEMS BE LIABLE FOR THE COST OF PROCUREMENT OF SUBSTITUTE GOODS.
Full details of R&D Systems' Terms and Conditions of Sale can be found online at: RnDSystems.com/Legal.
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