Qk006 recombinant human R-spondin 1 (RSPO1)
Protein quality information for batch #010
All our proteins are produced in-house by our scientists and we provide detailed quality data for each individual batch. Please contact us any time by email email@example.com or phone +44 (0) 1223 491486 if you have any questions.
After aliquotting and lyophilising the protein, 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 of each batch is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. We use a sensitive test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). Bioactivity of the protein is determined using the quantitative TOP-FLASH Wnt reporter assay. 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.
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.
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.
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.
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.
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.
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.