Recombinant human R-spondin 3 protein

£170.00£1,600.00

Qk032 Recombinant human R-spondin 3 protein is a 17 kDa bioactive fragment of human R-spondin 3 protein comprised of the two cysteine-rich furin-like domains, which are essential for its activity in stem cell and organoid culture. Our recombinant R-spondin 3 has been purified to homogeneity and is animal-free to ensure batch to batch consistency when adding to chemically-defined culture media.

Qk032 Recombinant human R-spondin 3 protein

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) 4-62. In the colon, R-spondin 3 promotes stem cell recovery and epithelial repair via induction of Wnt.

Qk032 Recombinant human R-spondin 3 protein is a biologically active protein fragment comprising the two cysteine-rich, furin-like domains, which are necessary and sufficient for Wnt signal potentiation and are the essential domains for activity in organoid culture.

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 organoids2. 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 labs3.

We produce our recombinant human R-spondin 3 protein in E.coli, so the protein is not glycosylated and is produced in an animal-derived component free laboratory making it ideal for formulation of defined media. The glycosylation of our recombinant human R-spondin 3 protein is unnecessary for extracellular signalling and stability. Our methods allow for optimized reliable production of homogeneous protein and enhanced comparability between batches. We provide bulk pricing and batch stock reservation for this protein.

Summary: bioactive domain of human R-spondin 3 protein (Uniprot: Q9BXY4)

Form: protein is provided lyophilized from a fully volatile solution without carrier protein.  Animal-derived component free. 

Molecular mass: ~17 kDa

Purity and bioactivity

For peace of mind that our recombinant human R-spondin 3 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, reversed-phase chromatography, UV spectroscopy and endotoxin level testing.

To confirm the biochemical identity of our recombinant human R-spondin 3 protein and ensure that its purity meets our rigorous standards, we conduct SDS-PAGE on every protein batch.

We confirm the consistent bioactivity of batches using enhancement of Wnt-3 signalling in the TOP-FLASH reporter 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 3 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

Recombinant human R-spondin-3 protein purity in SDS-PAGE

Bioactivity: R-spondin 3 enhancement of Wnt-3 signalling in TOP-FLASH luciferase reporter assay

Bioactivity of recombinant human R-spondin 3 protein in Wnt TOP FLASH assay

Research applications

Organoid culture

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.  Please contact us with questions any time by email at support@qkine.com or phone +44 (0) 1223 491486 / US toll free 1-866 877 2185.

Order online and upload your PO or pay by credit card,  or email your PO to orders@qkine.com.

We provide bulk orders and stock reservation for sensitive applications, please email us.

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 reversed-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 when you use the proteins you can rely on your calculated dilution.

Purity: SDS-PAGE

Result: R-spondin 3 bioactive domain migrates at 17 kDa in non-reducing (-βME) conditions and upon reduction (+βME).

Recombinant human R-spondin 3 protein purity in SDS-PAGE

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.

Bioactivity: enhancement of Wnt-3 signalling in TOP-FLASH luciferase reporter assay

Result: RSPO 3 enhances Wnt signalling in the TOP-FLASH reporter assay with EC50 = 0.49 nM

Bioactivity of recombinant human R-spondin 3 protein in Wnt TOP-FLASH assay

Bioactivity using a Wnt reporter assay in HEK293T cells. HEK293T cells transfected with Wnt-responsive firefly luciferase reporter TOPflash are treated with increasing concentrations of Qk032 R-spondin 3 #010 in the presence of Wnt-conditioned media (1:8 dilution), in triplicate. The following day, luciferase activity is measured by luminescence.

Purity: mass spec analysis

Purity: analytical reversed-phase chromatography

Result: Reversed-phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.

Purity and structural homogeneity of our recombinant human R-spondin 3 protein is analyzed by reversed-phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analyzed in ACE C4 4.6 x 250 mm column eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid. Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient. Example data from Qk001 batch #011

Protein purity and structural homogeneity is analyzed by reversed-phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analyzed in ACE C4 4.6 x 250 mm column eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid . Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient. Example data from Qk032 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.

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.

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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 human R-spondin 3 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 disulfide structure of the protein by minimizing 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 store frozen.
  • Our proteins 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 at -80°C for long-term storage or at -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 when you use the proteins you can rely on your calculated dilution.

Recovery: protein quantitation

Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.

Recovered concentration: 1.05 mg/ml or Recovery: 105%

Check full recovery from vial of recombinant human R-spondin 3 protein using UV spectra

The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. This was diluted 1:5 and the UV spectrum from 340-220 nm read. The concentration was calculated using the extinction coefficient at 280 nm. Example data from batch #010.

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