Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

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 (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).

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.

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).

• Reactivity: Mu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF Summary

• Additional Information: Widely used for both human and mouse cell culture.
• Details of Functionality: 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.
• 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.
• Accession #: Q62226.2
• N-terminal Sequence: Met-Ile-Ile-Gly26-Pro-Gly-Arg-Gly-Phe-Gly
• Protein/Peptide Type: GMP Recombinant Proteins
• Purity: >97%, by SDS-PAGE with quantitative densitometry by Coomassie® Blue Staining. The molecular weight by mass spectrometry is 19844 Da ± 26 Da
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 19.8 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: 21 kDa, under reducing conditions.

Packaging, Storage & Formulations

• 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.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Sodium Phosphate, NaCl and DTT with Trehalose.
• Purity: >97%, by SDS-PAGE with quantitative densitometry by Coomassie® Blue Staining. The molecular weight by mass spectrometry is 19844 Da ± 26 Da
• Reconstitution Instructions: Reconstitute at 500 μg/mL in sterile water.

Notes

Full terms and conditions of sale can be found online in the Protein Sciences Segment T&Cs at: Terms & Conditions.

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

Alternate Names for Recombinant Mouse Sonic Hedgehog/Shh GMP Protein, CF

• HHG1
• HHG-1
• HLP3
• HPE3
• MCOPCB5
• MCOPCB5sonic hedgehog (Drosophila) homolog
• Shh
• ShhNC
• SMMCI
• SMMCIsonic hedgehog homolog (Drosophila)
• sonic hedgehog homolog
• sonic hedgehog protein
• Sonic Hedgehog
• TPT
• TPTPS

Background

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).

1. Briscoe, J. and P.P. Therond (2013) Mol. Cell. Biol. 14:416.
2. Aviles, E.C. et al. (2013) Front. Cell. Neurosci. 7:86.
3. Xie, J. et al. (2013) OncoTargets Ther. 6:1425.
4. Echelard, Y. et al. (1993) Cell 75:1417.
5. Zeng, X. et al. (2001) Nature 411:716.
6. Feng, J. et al. (2004) Development 131:4357.
7. Goetz, J.A. et al. (2006) J. Biol. Chem. 281:4087.
8. Pepinsky, R.B. et al. (1998) J. Biol. Chem. 273:14037.
9. Chen, M.-H. et al. (2004) Genes Dev. 18:641.
10. Etheridge, L.A. et al. (2010) Development 137:133.
11. Jakobs, P. et al. (2014) J. Cell Sci. 127:1726.
12. Dierker, T. et al. (2009) J. Biol. Chem. 284:8013.
13. Lewis, P.M. et al. (2001) Cell 105:599.
14. Carpenter, D. et al. (1998) Proc. Natl. Acad. Sci. USA 95:13630.
15. Filmus, J. and M. Capurro (2014) Matrix Biol. 35:248.
16. Chuang, P.-T. and A.P. McMahon (1999) Nature 397:617.

Bioinformatics

• Uniprot:
  • Q62226.2()