Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF GMP

Name: Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF
Brand: R&D Systems
SKU: BT-SHH-GMP

R&D Systems, part of Bio-Techne | Catalog # BT-SHH-GMP

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Product Datasheet / COA / SDS

Widely used for both human and mouse cell culture.

Key Product Details

SHH Manufactured in Bio-Techne's new GMP facility
Lot-to-lot consistency
Stringent guidelines for patient safety
Scalability necessary to support successful therapeutics
Learn more about manufacturing in our new GMP facility
Test it in your process! Request a sample of GMP SHH

Source:	E. coli
Accession #:	Q62226.2
Applications:	Bioactivity

View Full Technical Details for Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

Technical Data
Citations / Reviews

Product Specifications for Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

Source	E. coli-derived mouse Sonic Hedgehog/Shh protein Cys25 - Gly198 (Cys25Ile-Ile) with an N-terminal Met Produced using non-animal reagents in an animal-free laboratory. Manufactured and tested under cGMP guidlines.
Purity	>97%, by SDS-PAGE with quantitative densitometry by Coomassie® Blue Staining. The molecular weight by mass spectrometry is 19844 Da ± 26 Da
Endotoxin level	<0.10 EU per 1 μg of the protein by the LAL method.
N-terminal sequence Analysis	Met-Ile-Ile-Gly26-Pro-Gly-Arg-Gly-Phe-Gly
Predicted Molecular Mass	19.8 kDa
SDS-PAGE	19.8 kDa
Activity	Measured by its ability to induce alkaline phosphatase production by C3H10T1/2 mouse embryonic fibroblast cells. Nakamura, T. et al. (1997) Biochem. Biophys. Res. Commun. 237:465. The ED₅₀ for this effect is 0.0500-0.600 μg/mL.

Scientific Data Examples for Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

	Recombinant Mouse Sonic Hedgehog/Shh GMP Protein Bioactivity. The bioactivity of Recombinant Mouse Sonic Hedgehog/Shh GMP Protein (Catalog # BT-SHH-GMP) was measured in the alkaline phosphatase production using C3H10T1/2 mouse embryonic fibroblast cells. Three independent lots were tested for bioactivity and plotted on the same graph to show lot-to-lot consistency of GMP Sonic Hedgehog/Shh protein.
	Equivalent Bioactivity of GMP and Animal-Free grades of Recombinant Mouse Sonic Hedgehog/Shh. Equivalent bioactivity of GMP (Catalog # BT-SHH-GMP) and Animal-Free (BT-SHH-AFL) grades of Recombinant Mouse Sonic Hedgehog/Shh as measured in the alkaline phosphatase production using C3H10T1/2 mouse embryonic fibroblast cells (orange and green, respectively).
	Recombinant Mouse Sonic Hedgehog/Shh GMP Protein SDS-PAGE. 2 μg/lane of Recombinant Mouse Sonic Hedgehog/Shh GMP Protein (Catalog # BT-SHH-GMP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 21 kDa.
	Recombinant Mouse Sonic Hedgehog/Shh GMP Protein Supports Stem Cell Differentiation to Dopaminergic Neurons. Representative immunofluorescent staining of cells for characteristic markers of neuronal lineage. Following differentiation using Recombinant Mouse Sonic Hedgehog/Shh GMP Protein (Catalog # BT-SHH-GMP), the iPSC-derived dopaminergic neurons were fixed and stained with DAPI (Catalog # 5748), anti-TH (Catalog # AF7566), and anti-TUJ1 (Catalog # MAB1195), followed by secondary antibody staining with anti-mouse IgG Alexa Fluor 488 conjugated antibody and anti-sheep IgG NorthernLights NL557-Conjugated Antibody (Catalog # NL010).

Formulation, Preparation and Storage

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

BT-SHH-GMP

Formulation	Lyophilized from a 0.2 μm filtered solution in Sodium Phosphate, NaCl and DTT with Trehalose.
Reconstitution	Reconstitute at 500 μg/mL in sterile water.
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. A minimum of 12 months when stored at ≤ -20 °C as supplied. Refer to lot specific COA for the Use by Date. 1 month, 2 to 8 °C under sterile conditions after reconstitution. 3 months, ≤ -20 °C under sterile conditions after reconstitution.

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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). Mouse Shh encodes a 437 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).

Item	Value
References	Briscoe, J. and P.P. Therond (2013) Mol. Cell. Biol. 14:416. Aviles, E.C. et al. (2013) Front. Cell. Neurosci. 7:86. Xie, J. et al. (2013) OncoTargets Ther. 6:1425. Echelard, Y. et al. (1993) Cell 75:1417. Zeng, X. et al. (2001) Nature 411:716. Feng, J. et al. (2004) Development 131:4357. Goetz, J.A. et al. (2006) J. Biol. Chem. 281:4087. Pepinsky, R.B. et al. (1998) J. Biol. Chem. 273:14037. 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. Dierker, T. et al. (2009) J. Biol. Chem. 284:8013. Lewis, P.M. et al. (2001) Cell 105:599. Carpenter, D. et al. (1998) Proc. Natl. Acad. Sci. USA 95:13630. Filmus, J. and M. Capurro (2014) Matrix Biol. 35:248. Chuang, P.-T. and A.P. McMahon (1999) Nature 397:617.
Entrez Gene IDs	6469 (Human); 20423 (Mouse)
Alternate Names	HHG-1, HHG1, HLP3, HPE3, MCOPCB5, MCOPCB5sonic hedgehog (Drosophila) homolog, SMMCI, SMMCIsonic hedgehog homolog (Drosophila), Shh, ShhNC, Sonic Hedgehog, TPT, TPTPS, sonic hedgehog homolog, sonic hedgehog protein

Manufacturing Specifications

GMP Proteins
R&D Systems, a Bio-Techne Brand's GMP proteins are produced according to relevant sections of the following documents: USP Chapter 1043, Ancillary Materials for Cell, Gene and Tissue-Engineered Products and Eu. Ph. 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products.

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, mass spectrometry, 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 <71>
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.

Production

All molecular biology procedures use animal-free media and dedicated labware.
Dedicated fermentors are utilized in committed animal-free areas.

Purification

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.

Quality Assurance

Low Endotoxin Level.
No impairment of biological activity.
High quality product obtained under stringent conditions.

Please read our complete Animal-Free Statement

Product Specific Notices for Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

END USER TERMS OF USE OF PRODUCT

The following terms are offered to you upon your acceptance of these End User Terms of Use of Product. By using this product, you indicate your acknowledgment and agreement to these End User Terms of Use of Product. If you do not agree to be bound by and comply with all of the provisions of these End User Terms of Use of Product, you should contact your supplier of the product and make arrangements to return the product.

We suggest you print and retain a copy of these End User Terms of Use of Product for your records.

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.

R&D Systems, Inc. has the right, at its sole discretion, to modify, add or remove any terms or conditions of these End User Terms of Use without notice or liability to you. Any changes to these End User Terms of Use are effective immediately following the printing of such changes on this product insert. The most recent version of these End User Terms of Use of Product may be found at: RnDSystems.com/Legal.

You agree to review these End User Terms of Use of Product to ensure any subsequent use by you of R&D Systems' GMP Products following changes to these End User Terms of Use of Product constitutes your acceptance of all such changes.

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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