Recombinant human R-spondin-1 protein (Qk006)
R-spondin-1 (RSPO-1) 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 , RSPO 1 is used extensively in adult stem cell-derived organoid culture, intestinal organoid culture, hematopoietic stem cell specification and other iPSC and culture systems1–5.
Qkine RSPO1 protein is a highly pure biologically active protein fragment comprising two cysteine-rich, furin-like domains, the essential domains for activity in stem cell culture. Our E.coli-produced RSPO 1 is not glycosylated, which ensures reliable production of homogeneous protein and enhanced comparability between batches. The glycosylation of R-spondin 1 is not necessary for extracellular signalling and stability 6.
Summary: Bioactive domain of human R-spondin 1 (Uniprot: Q2MKA7 expressed in E.coli and purified to homogeneity.
Form: protein is provided lyophilised from a fully volatile solution without carrier protein.
Molecular mass: 13 kDa
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R-spondin-1 replaces conditioned media in pancreatic tumor organoid culture.
Data from Dennis Plenker, Tuveson Lab, CSHL.
R-spondin 1 may be poorly soluble in physiological solutions. Please follow the handling guidance for lyophilized cytokines below to minimise loss of protein due to precipitation or adsorption to plastic. We advise storing the recombinant protein at very low pH to before dilution in cell culture media or final working solutions. Low pH will also assist in maintaining the correct disulfide structure of the protein by minimising disulfide 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 carrier protein of your choice such as BSA, HSA or gelatin to further minimise loss by adsorption.
- Store in -80°C for long term storage. -20°C for short-term.
Every effort is made to ensure samples are sterile however we recommend sterile filtering after dilution in media or the final working solution
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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.
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-ßcatenin signaling in HEK239T cells. Example data from batch #010.
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.
RSPO1 in 100 mM sodium phosphate pH 7.4 was analysed 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.
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
50 µg of RSPO1 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.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
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 in 6 M guanidine hydrochloride, 20 mM sodium phosphate pH 7.4 and the UV spectrum 340-220 nm. Concentration was calculated using extinction coefficient at 280 nm. Example data from batch #010.
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 optimise 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 batch #010.
1. A biological study establishing the endotoxin limit for in vitro proliferation of human mesenchymal stem cells (2017). Yusuke Nomura, Chie Fukui, Yuki Morishita, Yuji Haishima. Regenerative Therapy, 7, 45-51.
Qk006 batch #010, #011, #012
R-spondin-1 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.
All our proteins are produced in our Cambridge, UK, labs. We provide detailed quality data for each batch because we believe reliable, high quality cytokines and growth factors are critical for successful stem cell and organoid culture.
When we test our proteins, we choose a vial at random and reconstitute as recommended to ensure we are testing as close to the protein you will receive as possible. Biochemical identity and purity is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. Bioactivity is determined 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 when you use the proteins you can rely on your calculated dilution.
Please contact us with questions any time by email email@example.com or phone +44 (0) 1223 491486 / US toll free 1-866 877 2185.