Activin A plus 2.1 (Qk005)
Activin A is a member of the TGFβ superfamily of growth factors involved in stem cell differentiation and maintenance, regulation of embryogenesis, development of the reproductive system, wound healing and regulation of immune responses. The activity of Activin A is regulated by the high-affinity inhibitor, follistatin (1), and inhibins.
Activins are disulphide-linked homo- and heterodimers of four inhibin β chains. The best characterised are Activin A and Activin B, homodimers of inhibin βA and inhibin βB respectively. Activins, like all other members in the TGF-β superfamily, are synthesized as larger precursors consisting of an N-terminal signal peptide, a pro-domain of 250–350 residues and a highly conserved mature domain. The pro-domain, which is cleaved off in the mature protein, has important roles in the biosynthesis, stabilisation, transportation and signalling of the growth factors (2).
Activin A is used for maintenance of pluripotency in many human induced-pluripotent stem cell and human embryonic stem cell lines (3), and to induce stem cell differentiation into endoderm (4) and other cell fates.
Summary: Qk005 engineered mature domain of human activin A (Uniprot: P08476) expressed in E.coli, refolded and purified to homogeneity. This engineered form of Activin A incorporates an N-terminal truncation to remove a disulphide-linked extension. The resulting protein is optimised for extremely consistent high yield with no observed changes in bioactivity compared to the wild-type. This form is particularly suitable for large scale stem cell culture processes. The mature engineered protein is a disulphide-linked dimer.
Form: protein is provided lyophilised from a fully volatile solution without carrier protein.
Reconstitution instructions: Activin A 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: ~24 kDa (for the dimer)
1. Harrington, A. E. et al. Structural basis for the inhibition of activin signalling by follistatin. EMBO J. 25, 1035–1045 (2006).
2. Wang, X., Fischer, G. & Hyvönen, M. Structure and activation of pro-activin A. (2016). doi:10.1038/ncomms12052
3. Pauklin, S. & Vallier, L. Activin/Nodal signalling in stem cells. Development 142, 607–19 (2015).
4. D’Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).
Result: ActA+ 2.1 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. Data from Qk005 batch #010.
Result: ActA 2.1 activity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. EC50 = 5.6 pM . EC50 is within the expected range of 6 ± 2 pM.
Bioactivity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. Cells are treated (in triplicate) with a serial dilution of activin A for 6 hours. Firefly luciferase activity is measured and normalised to the control Renilla luciferase activity. Data from Qk005 batch #010.
Result: calculated molecular mass of the Activin A dimer is 24365.82 Da. Result of the analysis:24382.0 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. Data from Qk005 batch #010.
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. Data from Qk005 batch #010.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
Absorbance at 280 nm: average 0.180
Recovered concentration: 0.180 cm-1 x 10 / 0.76 cm-1 mg ml-1 = 1.18 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. Data from Qk005 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. Data from Qk005 batch #010.
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.
View full batch quality testing data for Qk001