Recombinant Human SCF GMP Protein, CF

Recombinant Human SCF GMP Protein (Catalog # BT-SCF-GMP) as measured in a cell proliferation assay using TF-1 human erythroleukemic cells. Three independent lots were tested for activity and plotted on the same graph to show lot-to-lot consistency of Recombinant Human SCF GMP Protein.

Equivalent bioactivity of GMP (Catalog # BT-SCF-GMP) and Animal-Free (BT-SCF-AFL) grades of Recombinant Human SCF as measured in a cell proliferation assay using TF-1 human erythroleukemic cells (orange and green, respectively).

2 μg/lane of Recombinant Human SCF/c‑kit Ligand GMP Protein (Catalog # BT-SCF-GMP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 18 kDa.

Three independent lots of Recombinant Human SCF GMP Protein (Catalog # BT-SCF-GMP) were analyzed by Maurice CE-SDS PLUS (IS is an Internal Standard). A gel-like representation of the purity analysis data (inset) can be obtained from the Lane View feature in Compass software for iCE. Profiles from the three runs were superimposed, showing excellent manufacturing consistency.

Three independent lots of Recombinant Human SCF GMP Protein (Catalog # BT-SCF-GMP) were analyzed by Maurice icIEF using native fluorescence detection (Mkr 5.85 and 9.99 are pI Markers). Profiles from the three runs were superimposed, showing excellent manufacturing consistency.

Purified CD34+ cells derived from plerixafor mobilized peripheral blood were cultured in commercially available media. The media was supplemented with SCF (100 ng/mL, Catalog # BT-SCF-GMP), TPO (100 ng/mL, Catalog # BT-TPO-GMP), FLT-3L (100 ng/mL, Catalog # BT-FT3L-GMP), and SR1 (1 µM, Catalog # 7086). After 7 days, fold expansion was calculated from total viable cell counts. Frequency (%) of CD34+ cells was analyzed by flow cytometry on Days 0 and 7. The average fold expansion of n=2 technical replicates is shown.

Purified CD34+ cells derived from plerixafor mobilized peripheral blood were cultured in commercially available media. The media was supplemented with SCF (Catalog # BT-SCF-GMP, 100 ng/mL), TPO (Catalog # BT-TPO-GMP, 100 ng/mL), FLT-3L (Catalog # BT-FT3L-GMP, 100 ng/mL), and SR1 (Catalog # 7086, 1 µM). After 7 days, fold expansion was calculated from total viable cell counts. Percent (%) CD34+ cells was analyzed by flow cytometry on Days 0 and 7. The average fold expansion of n=3 technical replicates is shown.

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

Recombinant Human SCF GMP Protein, CF Summary

• Details of Functionality: Measured in a cell proliferation assay using TF-1 human erythroleukemic cells. Kitamura, T. et al. (1989) J. Cell Physiol. 140:323. The ED 50 for this effect is 1.00-8.00 ng/mL. The specific activity of recombinant human SCF is >1.0 x 10 6 units/mg, which is calibrated against the human SCF reference standard (NIBSC code: 91/682).
• Source: E. coli-derived human SCF/c-kit Ligand protein Glu26-Ala189, with a N-terminal Met. Produced using non-animal reagents in an animal-free laboratory. Manufactured and tested under cGMP guidelines.
• Accession #: P21583.1
• N-terminal Sequence: Met-Glu26-Gly-Ile-(Cys)-Arg-Asn-Arg-Val-Thr
• 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 18573 Da ± 50 Da
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 19 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: 18 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 degreesC as supplied. Refer to lot specific COA for the Use by Date. 1 month, 2 to 8 degreesC under sterile conditions after reconstitution. 3 months, ≤ -20 degreesC under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose.
• Purity: >97%, by SDS-PAGE with quantitative densitometry by Coomassie® Blue Staining. The molecular weight by mass spectrometry is 18573 Da ± 50 Da
• Reconstitution Instructions: Reconstitute at 500 μg/mL in PBS.

Notes

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Alternate Names for Recombinant Human SCF GMP Protein, CF

• c-kit Ligand
• DCUA
• DFNA69
• DKFZp686F2250
• familial progressive hyperpigmentation 2
• FPH2
• FPHH
• KIT ligand
• Kitl
• KITLG
• KL-1
• Mast cell growth factor
• MGF
• MGFSHEP7
• SCF
• SCFStem cell factor
• SFc-Kit ligand
• SHEP7
• SLF
• steel factor

Background

Stem cell factor (SCF), also known as c-kit ligand (KL), mast cell growth factor (MGF), and steel factor (SLF), is a widely expressed 28-40 kDa type I transmembrane glycoprotein (1). It promotes the survival, differentiation, and mobilization of multiple cell types including myeloid, erythroid, megakaryocytic, lymphoid, germ cell, and melanocyte progenitors (1-7). SCF is a primary growth and activation factor for mast cells and eosinophils (8). Mature human SCF consists of a 189 amino acid (aa) extracellular domain (ECD), a 23 aa transmembrane segment, and a 36 aa cytoplasmic tail (9). The ECD shows both N-linked and O-linked glycosylation (10). Proteolytic cleavage at two alternate sites in the extracellular juxtamembrane region releases a 25 kDa soluble molecule which is comparable to the only form produced by Steel-dickie mutant mice (11, 12). An alternately spliced isoform of human SCF lacks 28 aa that encompasses the primary proteolytic recognition site (13). Within the ECD of the long isoform (corresponding to this recombinant protein), human SCF shares 79%-87% aa sequence identity with canine, feline, mouse, and rat SCF. Rat SCF is active on mouse and human cells, but human SCF is only weakly active on mouse cells (9). Noncovalent dimers of transmembrane or soluble SCF interact with the receptor tyrosine kinase SCF R/c-kit to trigger receptor dimerization and signaling (14). SCF assists in the recovery of cardiac function following myocardial infarction by increasing the number of cardiomyocytes and vascular channels (15). SCF is a versatile factor in the differentiation of many specific cell types like spermatogonial stem cells (16) and megakaryocyte progenitors (17). Apart from differentiation, SCF also can maintain stemness in cells. This is the case for human bone marrow mesenchymal cells, which require SCF and hepatocyte growth factor for maintenance (18). Hematopoietic stem cells similarly require SCF from surrounding cells in their niche to maintain their stemness and their progenitors (19). SCF has also improved protocols for continuous generation of cells in culture systems, like granulocytes and macrophages (20). For treatment of graft versus host disease, SCF is used in combination with other cytokines to generate myeloid-derived suppressor cells from human umbilical cord blood (21). SCF is also used to generate T cells for cell-based therapies, drug screening and disease modeling (22). In regenerative studies, SCF is applied in wound healing hydrogel as a means of increasing its adhesion strength and tissue regeneration (23).

1. Ashman, L.K. (1999) Int. J. Biochem. Cell Biol. 31:1037. 
2. Sette, C. et al. (2000) Int. J. Dev. Biol. 44:599. 
3. Yoshida, H. et al. (2001) J. Invest. Dermatol. Symp. Proc. 6:1. 
4. Erlandsson, A. et al. (2004) Exp. Cell Res. 301:201. 
5. Kapur, R. et al. (2002) Blood 100:1287. 
6. Wang, C.-H. et al. (2007) Arterioscler. Thromb. Vasc. Biol. 27:540. 
7. Bashamboo, A. et al. (2006) J. Cell Sci. 119:3039. 
8. Reber, L. et al. (2006) Eur. J. Pharmacol. 533:327.
9. Martin, F.H. et al. (1990) Cell 63:203.
10. Arakawa, T. et al. (1991) J. Biol. Chem. 266:18942.
11. Majumdar, M.K. et al. (1994) J. Biol. Chem. 269:1237.
12. Brannan, C.I. et al. (1991) Proc. Natl. Acad. Sci. 88:4671.
13. Anderson, D.M. et al. (1991) Cell Growth Differ. 2:373.
14. Lemmon, M.A. et al. (1997) J. Biol. Chem. 272:6311.
15. Kanellakis, P. et al. (2006) Cardiovasc. Res. 70:117.
    
16. Nasimi, M. et al. (2021) Reprod Sci. 28:963.
17. Krisch, L. et al. (2021) Int. J. Mol. Sci. 22:8224.
18. Cao, Z. et al. (2020) Stem Cell Res Ther. 11:1.
19. Comazzetto, S. et al. (2019) Cell Stem Cell. 24:477.
20. Bernecker, C. et al. (2019) Stem Cells Dev. 28:1540.
21. Park, M.Y. et al. (2019) Front Immunol. 10:1.
22. Netsrithong, R. et al. (2020) Stem Cell Res Ther. 11:1.
23. Zhang, Li. et al. (2021) Journal Mater Chem B. 29:5887. 

Bioinformatics

• Uniprot:
  • P21583.1()