Recombinant human Gremlin 1 protein (Qk015)

£93.00£696.00

Recombinant human Gremlin 1 protein has been optimized by our experts for exceptionally high-purity production and bioactivity.   Gremlin-1 is a BMP-inhibitor present in the natural intestinal niche and provides an animal-free substitute for Noggin in chemically-defined media for many ESC, iPSC and organoid culture systems. 

Qk015 Recombinant human Gremlin 1 protein

Gremlin 1 protein (GREM1, isoform-1) belongs to the BMP (bone morphogenetic protein) antagonist family. Gremlin 1 protein binds BMP2, BMP4, BMP7 and other BMP family proteins1 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 cells2. Expression of Gremlin 1 protein is also detected in fetal brain and colon, and at lower levels in adult brain, prostate, pancreas and skeletal muscle.

Recombinant human Gremlin 1 protein 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. However, roles for recombinant Gremlin 1 protein in cancer stem cell maintenance in glioblastoma have been suggested3.

Qk015 recombinant human Gremlin 1 protein is optimized 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. Recombinant human Gremlin-1 protein can also substitute for Noggin in several embryonic and induced-pluripotent stem cell and organoid culture systems.

Summary: human Gremlin 1 protein (Uniprot: O60565) expressed in E.coli and purified to homogeneity.

Molecular mass: 18 kDa

Form: protein is provided lyophilized from a fully volatile solution and is carrier protein-free. All our proteins are animal-derived component free making them particularly suitable for applications requiring a chemically-defined media.

Purity and bioactivity

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

To confirm the biochemical identity of our recombinant human Gremlin 1 protein and ensure that its purity meets our rigorous standards, we conduct SDS-PAGE on every protein batch.  We also check the final refolded protein is bioactive by quantitative determination of inhibition of BMP protein activity in a BRE-HEK 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 every preparation of our recombinant human Gremlin 1 protein.

Find out more about our extensive purity testing in the next tab.

Example data from batch #011

Protein purity: SDS-PAGE in reduced and non-reduced conditions

Recombinant human Gremlin 1 protein purity in SDS-PAGE

Bioactivity: BRE-HEK293 luciferase reporter assay

Recombinant human Gremlin 1 protein bioactivity in a BRE-HEK293 luciferase reporter assay

Research applications

iPSC/ESC maintenance

iPSC/ESC differentiation

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 of our recombinant human Gremlin 1 protein 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 you can rely on your calculated dilution.

Purity: SDS-PAGE

Result: Gremlin 1 protein 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 the dissociation of the dimer during electrophoresis which gives the characteristic diffuse band.

Recombinant human Gremlin 1 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.  Data from batch #011.

Bioactivity: BRE-HEK293 luciferase reporter assay

Result: Gremlin-1 inhibits BMP-2 induced luciferase activity with an IC50 = 34 ng/ml (1.88 nM)

Recombinant human Gremlin 1 protein bioactivity in a BRE-HEK293 luciferase reporter assay

Gremlin 1 activity 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. IC50 = 34 ng/ml. Cells are treated (n=4) with a serial dilution of Gremlin 1 in BMP2 for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. Data are from Qk015 batch #011.

Purity: mass spec analysis

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 disulfide-linked).  There are no minor contaminants.

Recombinant human Gremlin 1 protein has the expected molecular weight in MALDI mass spectrometry analysis

Gremlin 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 #011.

Purity: analytical reversed-phase chromatography

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

Recombinant human Gremlin 1 protein shows a single sharp peak in analytical reversed-phase chromatography

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 % trifluoroacetic acid in 65 minutes. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient. Example data from batch #011.

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

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.

Gremlin 1 protein may be poorly soluble in physiological solutions. Please follow the handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing our recombinant human Gremlin 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 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 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.

Every effort is made to ensure samples are sterile; however, we recommend sterile filtering after dilution in media or the final working solution.

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

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

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)

Recovery of recombinant human Gremlin 1 protein

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.

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