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.
Recombinant human Gremlin 1 protein (Qk015)
£93.00 – £696.00
Qk015 Recombinant human Gremlin 1 protein
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.
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.
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)
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.
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.
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
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)
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.