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recombinant human R-spondin 1 protein (Qk006)
Human R-spondin 1 protein (RSPO1) is the prototypic member of the R-spondin family and is used to potentiate Wnt signalling in many organoid culture systems including intestinal and tumor (cancer) organoid culture. R-spondin 1 is also required for hematopoietic stem cell specification and cancer cell migration and survival.
13kDa highly pure, bioactive domain of human R-spondin 1 comprising the two cysteine-rich furin-like domains of R-spondin 1, which are necessary and sufficient for Wnt signalling potentiation and are the essential domains for activity in stem cell and organoid culture. Qk006 is animal-free and not glycosylated to ensure production of homogeneous protein and enhanced comparability between batches. Used to replace R-spondin conditioned media for improved reproducibility in chemically defined organoid culture media.
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1mg may be despatched as 2 x 500µg
summary
high purity human R-spondin 1 protein (Uniprot: Q2MKA7)
13 kDa
expressed in E. coli
animal-free (AOF) and carrier protein-free.
manufactured in our Cambridge, UK laboratories
lyophilized from acetonitrile, TFA
resuspend in 10mM HCl at >100 µg/ml, prepare single use aliquots, add carrier protein if desired and store frozen at -20 oC or -80 oC
featured applications
pancreatic tumor organoid culture
maintenance of urine-derived stem cells
1. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
2. de Lau, W., Peng, W. C., Gros, P. & Clevers, H. The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 28, 305–316 (2014).
3. Genthe, J. R. & Clements, W. K. R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways. Development 144, 590–600 (2017).
4. Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724–1743 (2016).
5. Broutier, L. et al. Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nat. Med. 23, 1424–1435 (2017).
6. Chang, C.-F. et al. N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Binding Ability. doi:10.3390/ijms17060937.
species similarity:
porcine – 98%
mouse – 96%
rat – 96%
bovine – 94%
alternative
R-spondin 1 LR5 protein (Qk031): a specialized form of R-spondin 1 that specifically binds to the LGR5 receptor, developed in the lab of Marc de la Roche (University of Cambridge).
bioactivity
RSPO 1 enhances Wnt signalling in TOP-Flash reporter assay in Wnt reporter assay. HEK293T cells transfected with Wnt-responsive firefly luciferase reporter TOP-FLASH are treated with increasing concentration of Qk006 R-spondin 1 #010 (diluted in DMEM with 0.5 % of FCS), in the presence of Wnt-conditioned media (1:8 dilution), in triplicate. Cells are grown overnight and luciferase activity measured by luminescence. RSP1 enhances Wnt-ß catenin signaling in HEK239T cells. EC50 = 72.4ng/ml (5.8 nM). Data from lot #104277.
purity
RSPO1 migrates as a single band at 16 kDa in non-reducing (NR) and 13 kDa in reducing (R) conditions.
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 Qk006 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
Human R-spondin 1 (RSPO-1) protein is a secreted activator of the canonical Wnt signaling pathway. A ligand for the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4-61 , recombinant R-spondin 1 protein is used extensively in adult stem cell-derived organoid culture, intestinal organoid culture, hematopoietic stem cell specification and other iPSC and culture systems1–5.
Qk006 recombinant human R-spondin 1 protein is a highly pure biologically active fragment comprising two cysteine-rich, furin-like domains – the essential domains for activity in stem cell culture. Our E. coli-produced recombinant R-spondin 1 protein is not glycosylated, which ensures reliable production of homogeneous protein and enhanced comparability between batches. The glycosylation of R-spondin 1 protein is not necessary for extracellular signalling and stability 6.
additional resources
Technote: R-spondin 1 in pancreatic organoids
Qkine recombinant human R-spondin 1 protein replaces conditioned media in pancreatic tumor organoid culture – data from Tuveson Laboratory, Cold Spring Harbor Lab
Comparison of pancreatic organoid growth over three passages.
P-0 indicates the initial start of the culture in full growth media supplemented with Wnt3a and R-spondin 1-conditioned media. ‘Rec. R-spondin 1’ culture conditions were created by depleting full growth media of R-spondin 1-conditioned media and supplementing it with Qk006 recombinant R-spondin 1. ‘No R-spondin 1’ culture conditions were created by depleting full growth media of R-spondin 1-conditioned media but not subsequently supplementing the media with an alternative source of R-spondin 1. No differences in organoid growth have been observed when using recombinant R-spondin 1 instead of R-spondin 1-conditioned media. Experiments have been conducted by Dennis Plenker, Ph.D. in the lab of Dr David Tuveson at Cold Spring Harbor Laboratory.
Our products are for research use only and not for diagnostic or therapeutic use. Products are not for resale.
Recombinant R-spondin-1 replaces R-spondin-1 conditioned media in pancreatic tumor organoid culture
Dennis Plenker, Tuveson Lab, CSHL –
Comparison of pancreatic organoid growth over three passages.
P-0 indicates the initial start of the culture in full growth media supplemented with Wnt3a and R-spondin-1-conditioned media. For the recombinant (rec.) R-spondin-1 and no R-spondin-1 conditions full growth media was depleted of R-spondin-1 conditioned media and supplemented with rec. R-spondin-1 or without R-spondin-1. No differences in organoid growth have been observed when using recombinant R-spondin-1 instead of R-spondin-1-conditioned media. Experiments have been conducted by Dennis Plenker, Ph.D. in the lab of Dr David Tuveson at Cold Spring Harbor Laboratory.
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Qkine R-spondin 1 treatment on urine-derived stem cells maintains cellular morphology and nuclear expression of the nephron progenitor marker, SIX2
Lisa Nguyen, Adjaye Lab, University of Dusseldorf –
Previous research suggests that the removal of R-spondin 1 from the culture media of urine-derived stem cells leads to loss of kidney progenitor molecules in mice. Therefore, it was hypothesized that the addition of R-spondin 1 to the culture media would result in maintenance of SIX2 progenitor molecules and thus maintenance of the undifferentiated state.
Urine-derived stem cells were treated with Qk006 R-spondin 1 at a concentration of 200 ng/ml for 5 days. The cells were then fixed and stained for SIX2.
Qk006 R-spondin 1 maintains urine-derived stem cell morphology
Urine-derived stem cells typically have a small, rice grain-like morphology and express nuclear SIX2. Comparing the morphology of untreated vs treated cells showed that cells treated with Qk006 R-spondin 1 had a similar morphology to the control, indicating that R-spondin 1 treatment maintains the undifferentiated state.
Qk006 maintains nuclear SIX2 expression in urine-derived stem cells
In urine-derived stem cells, SIX2 expression is predominately found in the cell nuclei, as shown in the untreated control. Cytoplasmatic expression of SIX2 was also observed, which indicates cell differentiation. Addition of Qk006 R-spondin 1 maintained nuclear SIX2 expression (as shown by immunofluorescence staining for SIX2) and therefore appears to keep the urine-derived stem cells in an undifferentiated state.
Lisa Nguyen (MSc) and team in the laboratory of Professor James Adjaye at the Institute for Stem Cell Research and Regenerative Medicine, University of Dusseldorf.
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