Qk005 Recombinant human Activin A Plus protein is a 24 kDa biologically active variant of the mature domain of human Activin A protein. Our recombinant Activin A Plus has been extensively optimized for manufacture at extremely high yields without affecting its bioactivity to support large-scale stem cell culture applications.
Recombinant human Activin A Plus protein
£180.00 – £1,900.00
Activin A is a member of the TGFβ superfamily of growth factors used in stem cell differentiation and maintenance. Activin A Plus, an engineered optimized form of Activin A, incorporates an N-terminal truncation to remove a disulfide-linked extension. The resulting protein can be manufactured at 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.
Activin A is involved in regulation of embryogenesis, development of the reproductive system, wound healing and regulation of immune responses in vivo. 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 characterized 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, stabilization, transportation and signalling of the growth factors (2).
Summary: Optimized mature domain of human activin A (Uniprot: P08476) – particularly suitable for bulk and scale-up stem cell culture applications
Molecular mass: ~24 kDa (for the dimer)
Form: protein is provided lyophilized from a fully volatile solution. Animal-derived component and carrier protein-free.
ActA, Activin beta-A chain, Erythroid differentiation protein (EDP), Follicle-Stimulating Hormone (FSH) releasing protein (FRP), INHBA, Inhibin beta A, Inhibin beta 1
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).
Purity and bioactivity
For peace of mind that our recombinant human Activin A Plus 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, reverse phase chromatography, UV spectroscopy and endotoxin level testing.
To confirm the biochemical identity of our recombinant human Activin A Plus protein and ensure that its purity meets are rigorous standards, we conduct SDS-PAGE on every protein batch. As Activin A is a disulfide-linked dimer, we use both reduced and non-reduced conditions, as well as appropriate stains, for our SDS-PAGE analysis.
We also check the final refolded protein is bioactive using an activin-responsive firefly luciferase 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 the preparation.
Find out more about our extensive purity testing in the next tab.
Example data from batch #010
Protein purity: SDS-PAGE in reduced and non-reduced conditions
Bioactivity: Activin A responsive luciferase reporter assay
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 is determined using SDS-PAGE, mass spectrometry and analytical reverse 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 when you use the proteins you can rely on your calculated dilution.
Result: ActA Plus 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.
Bioactivity: luciferase reporter assay
Result: ActA Plus 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 normalized to the control Renilla luciferase activity. Data from Qk005 batch #010.
Purity: mass spec analysis
Result: calculated molecular mass of the Activin A Plus 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.
Purity: analytical reverse phase chromatography
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Protein purity and structural homogeneity is analyzed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analyzed 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.
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.
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.
Pure Activin A Plus protein is very 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 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 minimizing 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 minimize loss by adsorption.
- Store in -80°C for long term storage. -20°C for short-term.
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 when you use the proteins 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 0.180
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.