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Qk032 R-spondin 3 (human) Qkine protein vial
Human R-spondin 3 Qk032 protein purity SDS-PAGE lot #010
RSPO3 R-spondin 3 Bioactivity graph

Recombinant human R-spondin 3 protein (Qk032)

Human R-spondin 3 protein potentiates Wnt signalling in and has been shown to function in crypt regeneration in the intestine and control stem cell and progenitor cell behaviour during kidney development. R-spondin 3 is used alongside R-spondin 1 in intestinal organoid culture systems.

Qk032 is a 17 kDa highly pure, bioactive domain of human R-spondin 3 comprising the two cysteine-rich furin-like domains, which are necessary and sufficient for Wnt signalling potentiation and are the essential domains for activity in stem cell and organoid culture. Animal origin-free and carrier-protein free.

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1000µg will be despatched as 2 x 500µg

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Summary

  • High purity bioactive domain of human R-spondin 3 protein (Uniprot: Q9BXY4)

  • >98%, by SDS-PAGE quantitative densitometry

  • 17 kDa

  • Expressed in E. coli

  • Animal origin-free (AOF) and carrier protein-free.

  • Manufactured in our Cambridge, UK laboratories

  • Lyophilized from acetonitrile, TFA

  • Resuspend in 10mM HCl at >100 µg/ml (provided with protein and free of charge), prepare single use aliquots, add carrier protein if desired and store frozen at -20°C or -80°C

Featured applications

  • Chemically defined media for organoid culture

R-spondin-3, RSPO3, RSPO-3, Protein with TSP type-1 repeat, PWTSR, Roof plate-specific spondin-3, Thrombospondin type-1 domain-containing protein 2, THSD2

Human

Species similarity:
Bovine – 98%
Porcine – 98%
Mouse – 97%
Rat – 97%

Frequently used together

Recombinant human noggin protein

Bioactivity

Human R-spondin 3 Qk032 protein bioactivity lot #010

R-spondin 3 activity is determined using the Wnt-responsive firefly luciferase reporter assay. HEK293T cells transfected with reporter TOP-FLASH are treated in triplicate with increasing concentration of R-spondin 3 in the presence of Wnt-conditioned media (1:8 dilution). RSP1 enhances Wnt-ß catenin signaling in HEK239T cells. Cells are grown overnight, and luciferase activity is measured and normalized. EC50 = 0.3 nM (5 ng/ml). Data from Qk032 #010.

Purity

Human R-spondin 3 Qk032 protein purity SDS-PAGE lot #010

R-spondin 3 bioactive domain migrates at 17 kDa in non-reducing (-βME) conditions and upon reduction (+βME). Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250. Data from Qk032 lot #010

Further quality assays

  • Mass spectrometry: single species with expected mass

  • Analytical reversed-phase: single sharp peak

  • Endotoxin: <0.005 EU/μg protein (below level of detection)

  • Recovery from stock vial:  >95%

We are a company founded and run by scientists to provide a service and support innovation in stem cell biology and regenerative medicine.  All our products are exceptionally high purity, with complete characterisation and bioactivity analysis on every lot.

Protein background

R-Spondin 3 (RSPO3) protein belongs to the R-spondin family comprizing four structurally-related, secreted ligands (RSPO 1-4) [1]. All contain furin-like and thrombospondin structural domains. R-spondins are activators of the canonical Wnt signaling pathway. Receptors include the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-6 [2]. In the colon, R-spondin 3 promotes stem cell recovery and epithelial repair via induction of Wnt.

An R-spondin, such as R-spondin 3, is an essential component of Wnt 3a, R-spondin 3 (or 1,2), noggin (WRN) media used widely in propagation of intestinal organoids [2]. There is a drive towards improved reproducibility in stem cell and organoid culture. Conditioned media (spent media from cells that secrete growth factors such as Wnt and R-spondin, along with several other factors and waste products) is both challenging to manufacture consistently and conveniently, and a source of variability between labs [3].

[1] de Lau, W. B. M., Snel, B. & Clevers, H. C. The R-spondin protein family. Genome biology 13, 242 (2012). doi.org/10.1186/gb-2012-13-3-242

[2] Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). doi.org/10.1038/nature07935

[3] VanDussen, K. L., Sonnek, N. M. & Stappenbeck, T. S. L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams. Stem cell research 37, 101430 (2019). doi.org/10.1016/j.scr.2019.101430

Publications using recombinant human R-spondin 3 protein (Qk032)

Mass Generation and Long-term Expansion of Hepatobiliary Organoids from Adult Primary Human Hepatocytes.
Preprint on 11 June 2024 by Marsee, A., Richie, A., Myszczyszyn, A., et al.

Our products are for research use only and not for diagnostic or therapeutic use.  Products are not for resale.

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