Recombinant human ActA protein

£240.00£2,400.00

Qk001 Recombinant human Activin A protein is a 26 kDa disulfide-linked dimer composed of the mature domain of human Activin A protein. Our recombinant Activin A protein has been extensively validated and benchmarked against other suppliers’ for its reproducibility in stem cell culture.

Animal-derived component and carrier protein-free.

Qk001 Recombinant human Activin A protein

Human Activin A (ActA) protein 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 protein is regulated by the high-affinity inhibitor, follistatin1, and inhibins.

Activins are disulfide-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 factors2.

Recombinant Activin A is used for maintenance of pluripotency in many human induced-pluripotent stem cell and human embryonic stem cell lines3, and to induce stem cell differentiation into endoderm4 and other cell fates.

Our recombinant Activin A protein has been extensively validated and benchmarked with other suppliers’ proteins in stem cell culture and other assays. View the results of this analysis and a commentary by Qkine’s founder Marko Hyvönen.

Summary: Qk001 mature domain of human activin A protein expressed in E. coli (residues 311-426, Uniprot: P08476). The mature protein is a disulfide-linked dimer.

Molecular mass: ~26 kDa (for the dimer)

Form: Protein is provided lyophilized from a fully volatile solution. Animal-derived component and carrier protein-free.

Purity and bioactivity

For peace of mind that our recombinant Activin A 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 Activin A protein and ensure that its purity meets our 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 #011

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

Qk001 Recombinant human Activin A protein runs as a 26kDa dimer in non-reduced conditions

Bioactivity: Activin A responsive luciferase reporter assay

Qk001 Recombinant human Activin A protein shows high bioactivity in an Activin A-responsive 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 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 when you use the proteins you can rely on your calculated dilution.

Purity: SDS-PAGE

Result: ActA 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.

Qk001 Recombinant human Activin A protein runs as a 26kDa dimer in non-reduced conditions

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 Qk001 batch #011.

Bioactivity: luciferase reporter assay

Result: Qk001 Recombinant human Activin A protein activity is determined using an activin-responsive firefly luciferase reporter in HEK293T cells.  EC50 = 6.1 pM .  EC50 is within the expected range of 6 ± 2 pM.

Qk001 Recombinant human Activin A protein shows high bioactivity in an Activin A-responsive luciferase reporter assay

Purity: mass spec analysis

Result: calculated molecular mass of our Qk001 Recombinant human Activin A dimer is 25932 Da. Result of the analysis: 25934 Da which is consistent with the calculated mass.  No significant heterogeneity is present.

Qk001 Recombinant human Activin A protein has the expected molecular mass in MALDI mass spectrometry analysis

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 disulfide-linked. The different peaks represent different charge states of the protein.  Data from Qk001 batch #011.

Purity: analytical reversed-phase chromatography

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

Qk001 Recombinant human Activin A protein shows a single sharp peak in analytical reversed-phase chromatography analysis

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 and 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.  Example data from Qk001 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.

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What others are saying

  1. Stem cell researcher, University of Oxford, UK

    Qkine Activin A works nicely for human pluripotent stem cell differentiation to endoderm

    Stem cell researcher, University of Oxford, UK

    We are using Qkine Activin A and it works nicely for human pluripotent stem cell differentiation to endoderm. University of Oxford, UK.

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Pure recombinant human Activin A protein is very poorly soluble in physiological solutions. Please follow our handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic.  We advise storing our recombinant Activin A 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 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 0.158
Recovered concentration: 0.158 cm-1 x 10 / 1.446 cm-1 mg ml-1 = 1.09 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.  Example data from Qk001 batch #011.

Blackford SJI, Ng SS, Segal JM, King AJF, Austin AL, Kent D, Moore J, Sheldon M, Ilic D, Dhawan A, Mitry RR, Rashid ST: Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy.. In: Stem Cells Transl Med, 2018.

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