Qk006 Recombinant human R-spondin-1 protein is a highly pure, 13 kDa bioactive fragment of human R-spondin 1 composed of the cysteine-rich furin-like domains, which are essential for its activity in stem cell and organoid culture. Our recombinant human R-spondin 1 protein has been optimized for expression in E. coli and is not glycosylated to ensure production of homogeneous protein and enhanced comparability between batches.
Recombinant human R-spondin 1 protein
£170.00 – £1,600.00
Qk006 Recombinant human R-spondin-1 protein
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.
Summary: Bioactive domain of human R-spondin 1 protein (Uniprot: Q2MKA7 expressed in E. coli and purified to homogeneity.
Form: Protein is provided lyophilized from a fully volatile solution. Animal and carrier protein free.
Molecular mass: 13 kDa
roof plate-specific spondin 1, Rspo1, R-spondin 1, Cristin 3
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 Heparin Binding Ability. doi:10.3390/ijms17060937.
Purity and bioactivity
For peace of mind that our recombinant human R-spondin 1 protein will work exactly the same way every day, from batch to batch and at any scale you need, we conduct extensive purity and bioactivity analysis, including SDS-PAGE, mass spectrometry, reverse phase chromatography, UV spectroscopy and endotoxin level testing.
We confirm the final refolded protein has consistent bioactivity by determining enhancement of Wnt3a signalling in the TOP-FLASH assay in HEK293T cells. By knowing what the expected activity of the protein is and measuring calibrant alongside each batch of protein, we can use this bioassay to define a complete dose-response curve and check the EC50 value of each preparation of our recombinant human R-spondin 1 protein.
Find out more about our extensive purity testing in the next tab.
Example data from batch #010
Protein purity: SDS-PAGE in reduced and non-reduced conditions
Bioactivity: Wnt3 reporter (TOP-FLASH) assay enhancement reporter assay
Our products are for research use only and not for diagnostic or therapeutic use. Products are not for resale.
For final purity and activity tests on our proteins, we choose a vial at random and reconstitute as recommended. Biochemical identity and purity is determined using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. Bioactivity is quantified using an appropriate cell-based assay. As stem cells are sensitive to endotoxin levels, we use a high resolution test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). We also check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so you can rely on your calculated dilution.
Result: 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. Example data from batch #010.
Bioactivity: TOP-FLASH Wnt-3 reporter assay
Result: RSPO 1 enhances Wnt signalling in TOP-Flash reporter assay with EC50 = 27 nM
Bioactivity using a Wnt reporter assay in HEK293T cells. 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 in the presence of Wnt-conditioned media (1:8 dilution), in triplicate. Cells are grown over-night and luciferase activity measured by luminescence. RSP1 enhances Wnt-ß catenin signaling in HEK239T cells. Example data from batch #010.
Purity: mass spec analysis
Result: theoretical molecular weight: 12521 Da. Result of the analysis: 12522.2 Da confirming the molecular weight of active domain of R-spondin 1 (with the assumption that all the cysteines are disulphide-linked). There are no minor contaminants.
Our recombinant R-spondin 1 in 100 mM sodium phosphate pH 7.4 was analyzed by mass spec. The different peaks represent different charge states of the protein. These are used to calculate the mass of the protein, which is then compared to the calculated theoretical mass. Example data from batch #010.
Purity: analytical reverse phase chromatography
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
50 µg of our recombinant human R-spondin 1 protein (batch #010) was diluted in 10 mM HCl to 0.1 mg/ml and run in an analytical ACE C4 4.6 x 250 mm column at 1 ml/min and eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoro acetic acid in 65 minutes. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient. Example data from batch #010.
Purity: endotoxin level determination
Result: Endotoxin level <0.005 EU/ug protein (below level of detection)
Stem cell cultures are sensitive to endotoxins1, which can be present in media, serum and as a contaminant on plasticware. We optimize our protein production processes to ensure the lowest possible levels of endotoxin contamination. Our endotoxin pass criteria are set at the industry leading <0.1 EU per ug protein and we aim for <0.01 EU per ug protein. Endotoxin levels in our proteins are determined by an external expert microbiological testing services provider. Example data from Qk001 batch #011
Pure R-spondin 1 protein is very poorly soluble in physiological solutions. Please follow our handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing our recombinant R-spondin 1 protein at very low pH before dilution in cell culture media or your final working solution. Low pH will also assist in maintaining the correct disulphide structure of the protein by minimizing disulphide bond exchange reactions.
- Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilized cytokines in 10 mM HCl (1:1000 dilution of concentrated HCl) while keeping the protein concentration at 50 µg/ml or above, in order to avoid loss by adsorption to plasticware.
- To ensure you recover all of the protein, let the sample sit for a few minutes with the solubilization buffer at room temperature and pipette gently up and down (avoid foaming).
- Rinse the tube with some more 10 mM HCl and pool with the rest.
- The protein is tolerant of some freeze and thaw cycles, but as always with proteins, it is better to aliquot and stored frozen.
- Our protein are supplied carrier-protein free. If compatible with your work, add a carrier protein of your choice such as BSA, HSA or gelatin to further minimize loss by adsorption.
- Store in -80°C for long-term storage. -20°C for short-term storage.
We check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so you can rely on your calculated dilution.
Recovery: protein quantitation
Absorbance at 280 nm: average 0.05
Recovered concentration: 0.05 cm-1 x 10 / 0.436 cm-1 mg ml-1 = 1.14mg / ml
Recovery: 110% (>100% due to routine 10% over-fill of vials during aliquoting)
The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. This was diluted 1:10 and the UV spectrum 340-220 nm measured. Concentration was calculated using extinction coefficient at 280 nm. Example data from batch #010.
Our 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. 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.