Recombinant Gremlin 1 protein (Qk015)
Gremlin-1 (GREM1, isoform-1) belongs to the BMP (bone morphogenic protein) antagonist family. Gremlin-1 binds BMP2, BMP4, BMP7 and other BMP family proteins (1) and inhibits receptor binding. It is highly expressed in the small intestine at the base of the intestinal crypts, as are the related proteins, Gremlin 2 and Chordin-like 1. In this niche, they help maintain the stem cell population by inhibiting BMP produced by mesenchymal cells (2). Expression of Gremlin 1 is also detected in fetal brain and colon; and at lower levels in adult brain, prostate, pancreas and skeletal muscle.
Gremlin-1 and other BMP-antagonists such as Noggin are used in the derivation, growth and maintenance of organoids from epithelial tissues including intestinal, liver and pancreatic organoids. Roles in cancer stem cell maintenance in glioblastoma have been suggested (3).
Qkine recombinant Gremlin-1 protein is optimised for exceptionally high purity production from E.coli. All our proteins are animal-derived component free making them particularly suitable for applications requiring a chemically defined media. Gremlin-1 can substitute for Noggin in several embryonic and induced-pluripotent stem cell and organoid culture systems.
Summary: human Gremlin 1 (Uniprot: O60565) expressed in E.coli and purified to homogeneity.
Form: protein is provided lyophilized from a fully volatile solution and is carrier protein and animal-derived component free.
Molecular mass: 18 kDa
Reconstitution instructions: Gremlin-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 disulphide structure of the protein by minimising 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 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
1. Kišonaitė, M., Wang, X. & Hyvönen, M. Structure of Gremlin-1 and analysis of its interaction with BMP-2. The Biochemical journal 473, 1593–1604 (2016).
2. Kosinski, C. et al. Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proceedings of the National Academy of Sciences of the United States of America 104, 15418–15423 (2007).
3. Yan, K. et al. Glioma cancer stem cells secrete Gremlin1 to promote their maintenance within the tumor hierarchy. Genes & development 28, 1085–1100 (2014).
Result: Gremlin-1 migrates as a single diffuse band at ~36 kDa in non-reducing (NR) and 19 kDa in reducing (R) conditions. The protein is a non-covalent dimer and it is dissociation of the dimer during electrophoresis which gives the characteristic diffuse band.
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 batch #011.
Result: Gremlin-2 inhibits BMP-2 induced luciferase activity with an IC50 = 34 ng/ml (1.88 nM)
Gremlin1 activity is determined using a BMP2-responsive luciferase reporter assay in stably transfected HEK293T cells. IC50 = 34 ng/ml. Bioactivity is determined using inhibition of the BMP2 response (Qk007 #010, 52 ng/ml) from a BMP2-responsive firefly luciferase reporter in stably transfected HEK293T cells. Cells are treated (n=4) with a serial dilution of Gremlin 1 in BMP2 for 6 hours. Firefly luciferase activity is measured and normalised to the control Renilla luciferase activity. Data are from Qk015 batch #011.
Result: theoretical molecular weight: 18373 Da. Result of the analysis: 18380 Da confirming the molecular weight of Gremlin-1 (with the assumption that all the cysteines are disulphide-linked). There are no minor contaminants.
Gremlin-1 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 #011.
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
50 µg of Gremlin-1 batch #011 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 #011.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
Absorbance at 280 nm: average 0.058
Recovered concentration: 0.058 cm-1 x 10 / 1.84 cm-1 mg ml-1 = 1.06mg / ml
Recovery: 106% (>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.
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.
Qk015 batch #010, #011, #012
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 firstname.lastname@example.org or phone +44 (0) 1223 491486 if you have any questions.
After aliquotting and lyophilizing 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 a quantitative luciferase reporter assay. We also check that the correct amount of protein is recovered from the vial – it might sound simple but if you order 100 µg, we believe you should receive 100 µg.