Recombinant Human Sonic Hedgehog/Shh Protein, High Activity

C-terminal cholesterol, N-terminal fatty acid-modified
  
  • Purity
    >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
  • Endotoxin Level
    <0.10 EU per 1 μg of the protein by the LAL method.
  • 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 typically 6-36 ng/mL.
  • Source
    Human embryonic kidney cell, HEK293-derived Cys24-Gly197
  • Accession #
  • N-terminal Sequence
    Analysis
    Cys24
  • Predicted Molecular Mass
    20 kDa
  • SDS-PAGE
    18-24 kDa, reducing conditions
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.
8908-SH
 
8908-SH/CF
Formulation Supplied as a 0.2 μm filtered solution in MES, NaCl and CHAPS with BSA as a carrier protein.
Formulation Supplied as a 0.2 μm filtered solution in MES, NaCl and CHAPS.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Data Images
Enhanced Activity of Human Cell-expressed Sonic Hedgehog (Shh).
Recombinant Human Shh proteins induce alkaline phosphatase production by mesenchymal stem cells. High Activity Shh (green), purified from HEK293 cells and containing the correct post-translational modifications (cholesterol and fatty acids), is over 14-fold more active than E. coli-purified Recombinant Human Shh-N (C24II)
N-Terminus (Catalog # 1845-SH; red line), and over 250-fold more active than E. coli-purified Recombinant Human Shh-N (Catalog # 1314-SH; blue line).
1 μg/lane of Recombinant Human Sonic Hedgehog/Shh was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing single bands at 14 kDa and 12 kDa, respectively.
Post-translational Modification Analysis of Naturally-modified Recombinant Human Sonic Hedgehog (Shh).

LC/ESI-MS analysis of Recombinant Human (rh)SHH, High Activity shows major peaks at 20119.3, 20145.2, and 20171.6 Da, suggesting that recombinant human SHH molecules are dual-modified with cholesterol at C-terminus, and fatty acids (lauric acid, myristic acid, and palmitic acid) at the
N-terminus. The minor peaks at 19776 Da corresponds to rhSHH with only fatty acid modification.

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).
  • References:
    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. Marigo, V. et al. (1995) Genomics 28:44.
    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.
  • Entrez Gene IDs:
    6469 (Human); 20423 (Mouse)
  • Alternate Names:
    HHG1; HHG-1; HLP3; HPE3; MCOPCB5sonic hedgehog (Drosophila) homolog; Shh; SMMCIsonic hedgehog homolog (Drosophila); sonic hedgehog homolog; sonic hedgehog protein; sonic hedgehog; Sonic Hedgehog/Shh; TPT; TPTPS
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