GDF15 – human (Qk017)
Growth/differentiation factor 15 (GDF15) is a distant member of the TGFβ superfamily (Uniprot: Q99988). Its expression is tightly regulated and circulating GDF15 in serum is associated with diseases such as cancer, cardiovascular disease, obesity and metabolic disease.
Unlike other members of the TGFβ superfamily that cause activation of the SMAD pathway, GDF15 signals through GRAL and co-receptor RET leading to RET phosphorylation and signalling through the ERK and AKT pathway (reviewed in Emmerson et al., 2018). Commercial sources of GDF15, in particular those purified from mammalian expression are frequently contaminated with trace amounts of TGFβ and related proteins. These trace contaminants cause misleading experimental results due to the picomolar or even femtomolar EC50s of this family of cytokines (2).
We produce our proteins in E.coli with no animal products in our culture or purification processes to ensure there is no contamination from related proteins. In addition, we use a well-characterised SMAD2/3 activation assay to confirm there is no SMAD signalling.
Summary: Qk015 Mature domain of human GDF15 (Uniprot: Q99988) expressed in E.coli and purified to homogeneity. Mature active protein is a disulphide-linked dimer.
Form: protein is provided lyophilised from a fully volatile solution without carrier protein.
Reconstitution instructions: GDF15 is very poorly soluble in physiological solutions. Please follow the handling guidance for lyophilised 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 lyophilised 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 solubilisation 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
Molecular mass: ~25 kDa (for the dimer)
Result: GDF15 migrates as a single band at 24 kDa in non-reducing (NR) and 13 kDa as a single monomeric species upon reduction (R). No contaminating protein bands are visible.
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: calculated molecular mass of the GDF15 dimer is 24556.4 Da. Result of the analysis: 24556.8 Da which is consistent with the calculated mass. No significant heterogeneity is present.
MALDI mass spectrometric analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident in SDS-PAGE analysis. The results are compared with calculated mass of the protein with the assumption that all the cysteines are disulphide-linked. The different peaks represent different charge states of the protein.
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Protein purity and structural homogeneity is analysed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analysed in ACE C4 4.6 x 250 mm column using 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.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
Absorbance at 280 nm: average 0.13
Recovered concentration: 0.13 cm-1 x 10 / 1.17 cm-1 mg ml-1 = 1.1 mg / 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.
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.
GDF15 signals through GRAL and co-receptor RET leading to RET phosphorylation and signalling through the ERK and AKT pathway (reviewed in Emmerson et al., 2018). Commercial sources of GDF15, in particular those purified from mammalian expression systems have been shown previously to be contaminated with trace amount of TGFβ. These trace contaminants cause misleading experimental results due to the picomolar or even femtomolar EC50s (Olsen et al., 2017). Here we use a well characterised SMAD2/3 activation assay to show that there is no contamination from other TGFβ family proteins.
Result: EC50 = 0 pM (no contamination with TGFβ or related growth factors)
Bioactivity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. Cells are treated (in triplicate) with a serial dilution of GDF15 or Activin A for 6 hours. Firefly luciferase activity is measured and normalised to the control Renilla luciferase activity.
View full batch quality testing data for Qk017
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 activin responsive luciferase 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.