Recombinant Human EGF GMP Protein, CF Summary
The specific activity of Recombinant Human EGF is >8.0 x 105 IU/mg, which is calibrated against the human EGF WHO International Standard (NIBSC code: 91/530).
Asn971-Arg1023, with an N-terminal Met
Produced using non-animal reagents in an animal-free laboratory.
Manufactured and tested under cGMP 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.|
|Reconstitution||Reconstitute at 200 μg/mL in PBS.|
|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.
GMP-grade Recombinant Human EGF (Catalog # 236‑GMP) stimulates proliferation of the Balb/3T3 mouse embryonic fibroblast cell line. The ED50for this effect is 20‑100 pg/mL. Three independent lots were tested for activity and plotted on the same graph to show lot-to-lot consistency of GMP EGF.
1 µg/lane of GMP-grade Recombinant Human EGF (Catalog # 236-GMP) was resolved by SDS-PAGE and visualized by silver staining under reducing (R) conditions, showing a single band at 6 kDa.
MALDI‑TOF analysis of GMP‑grade Recombinant Human EGF (Catalog # 236‑GMP). The major peak corresponds to the calculated molecular mass, 6353 Da. The minor peak at 6549 Da is a matrix-associated artifact of the MALDI-TOF.
Epidermal growth factor (EGF) is a small, potent growth factor capable of inducing cell proliferation, differentiation, and survival. EGF is the founding member of the EGF family that also includes TGF-alpha, amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), heparin‑binding EGF‑like growth factor (HB‑EGF), epigen, and the neuregulins (NRG)-1 through -6 (1). Members of The EGF family are characterized by a shared structural motif, the EGF‑like domain, which contains three intramolecular disulfide bonds that are formed by six similarly spaced, conserved cysteine residues (2). These disulfide bonds are essential for proper protein conformation and receptor binding. All EGF family members are synthesized as type I transmembrane precursor proteins that may contain several EGF domains in the extracellular region. The mature proteins are released from the cell surface by regulated proteolysis (1). The full length EGF protein is 1207 amino acids (aa) (EGF precursor) containing nine EGF domains and nine LDLR class B repeats. However, the mature protein is much smaller, only 53 aa, and is generated by proteolytic cleavage of the EGF domain proximal to the transmembrane region (3). EGF is well conserved across mammals with mature human EGF 70% identical to mature mouse and rat EGF. Physiologically, EGF is found in various body fluids, including blood, milk, urine, saliva, seminal fluid, pancreatic juice, cerebrospinal fluid, and amniotic fluid (4). EGF is a high affinity ligand of the EGF receptor (ErbB). Four ErbB (HER) family receptor tyrosine kinases including EGFR/ErbB1, ErbB2, ErbB3 and ErbB4, mediate responses to EGF family members (5). EGF binding induces dimerization of the EGF receptor resulting in activation of the protein tyrosine kinase signaling pathway. These receptors undergo a complex pattern of ligand-induced homo- or hetero-dimerization to transduce EGF family signals (6, 7). EGF binds ErbB1 and depending on the context, induces the formation of homodimers or heterodimers containing ErbB2. Dimerization results in autophosphorylation of the receptor at specific tyrosine residues to create docking sites for a variety of signaling molecules (5, 8). Biological activities ascribed to EGF include epithelial development, angiogenesis, inhibition of gastric acid secretion, fibroblast proliferation, and colony formation of epidermal cells in culture.
- Harris, R.C. et al. (2003) Exp. Cell Res. 284:2.
- Carpenter, G. and Cohen, S. (1990) J. Biol. Chem. 265:7709.
- Bell, G.I. et al. (1986) Nucl. Acids Res. 14:8427.
- Carpenter, G. and Zendegui, J.G. (1986) Exp. Cell Res. 164:1.
- Jorissen, R.N. et al. (2003) Exp. Cell Res. 284:31.
- Gamett, D.C. et al. (1997) J. Biol. Chem. 272:12052.
- Qian, X. et al. (1994) Proc. Natl. Acad. Sci. 91:1500.
- Qian, X. et al. (1999) J. Biol. Chem. 274:574.
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
- N-terminal amino acid analysis, SDS-PAGE analysis, mass spectrometry results, 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
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.
The vial is supposed to contain lyophilized protein but it appears to be empty. Is there anything in it?
Pellets can be dislodged during shipping and become disbursed on the vial wall and in the cap. Centrifuge or tap the vial on the benchtop to return this material to the vial bottom. If this does not reveal a pellet, closely inspect the cone of the vial. Some pellets appear as only a tiny amount of material or as a transparent film due to the original buffer formulation. This is a normal appearance for many proteins. For example, if the product is originally lyophilized from a solvent such as acetonitrile or ethanol, and supplied carrier-free, you may not be able to detect the pellet with the naked eye. This does not mean the vial is empty. Reconstitute the vial as directed. After reconstitution, protein concentration can be tested with a spectrophotometer.
What is the recommended method for reconstitution of a lyophilized protein or antibody?
Unless more specific directions are on the Certificate of Analysis provided with the product, we suggest the following procedure to ensure optimal recovery: 1. Allow the vial and reconstitution buffer to equilibrate to room temperature. 2. Briefly centrifuge the vial to ensure that all lyophiliate is collected at the bottom of the vial. 3. Add the amount of buffer required to achieve the concentration recommended on the product insert. 4. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation, like on a rocker platform or rotating by hand. Avoid vigorous shaking that can cause foaming and protein denaturation. 5. Aliquot into volumes greater than 20 μL and store as indicated on the product insert. If the vial exhibits flakes or particulates, mix the product for a couple of hours at room temperature and then at 4oC overnight. Contact Technical Service if product does not go into solution.
Animal-Free Recombinant Proteins
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