Recombinant human TGF-β1 PLUS protein is the first entirely animal-free recombinant human transforming growth factor beta 1 (TGF-β1) protein for highly reproducible results and compatible with chemically-defined stem cell media. Our TGF-β1 PLUS protein has been extensively tested for maintenance of iPSC pluripotency by the specialist stem cell biotechnology company, Stemnovate, Cambridge, UK.
Recombinant human TGF-β1 PLUS protein (Qk010)
£116.00 – £6,960.00
- Purity & bioactivity data
- Customer & collaborator data
- Resuspension & storage
For final purity and activity tests on our proteins, we choose a vial at random and reconstitute as recommended. Biochemical identity and purity are 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.
Result: TGF-β1 PLUS (Qk010) dimer migrates as a single band at 24 kDa in non-reducing (NR) and 13 kDa as a single monomeric species upon reduction (R) with β-mercaptoethanol. High purity yield of dimeric protein (bioactive form).
Purified recombinant protein (7 μg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptoethanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250. Data from Qk010 batch #012.
Bioactivity: luciferase reporter assay
Result: TGF-β1 PLUS (Qk010) is highly bioactive compared to mammalian-expressed TGF-β1 preparations from other suppliers.
Comparative activity determined using a quantitative luciferase reporter assay in transiently transfected HEK293T cells. Cells were treated (in triplicate) with a serial dilution of TGF-β1. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. TGF-β1 PLUS EC50 1.4 pM. Suppliers 1 and 2 have EC50 ~3.5 pM and 38 pM respectively.
Purity: mass spec analysis
Result: Consistent with the calculated mass. No 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 disulfide-linked. The different peaks represent different charge states of the protein. Data from Qk010 batch #011.
Purity: endotoxin level determination
Result: Endotoxin level <0.005 EU/µg 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 Qk010 batch #011.
TGF-β1 PLUS maintains iPSC pluripotency with high Nanog expression
TGF-β1 PLUS is highly effective at maintaining iPSC pluripotency in chemically-defined, serum and feeder-free culture
Immuno-staining for pluripotency markers Tra 1-60 and Nanog show high levels of expression in iPSC lines maintained in TGF-β1 PLUS (Qk010)-containing defined media. In this study, Qkine TGF-β1 PLUS performed better than the TGF-β1 used routinely by Stemnovate.
TGF-β1 PLUS (Qk010) maintains pluripotency and good colony morphology at 1 ng/ml in a chemically-defined serum and feeder-free iPSC culture. TGF-β1 PLUS used at 1 ng/ml; Qk025 FGF2 used at 100 ng/ml.
TGF-β1 PLUS in combination with Qkine FGF2 shows enhanced bioactivity when benchmarked against other suppliers
Stemnovate IPS media (chemically-defined, serum and feeder-free culture). Higher Nanog pluripotency marker immuno-staining in human iPSC cultured in Qkine FGF2 (Qk025) and TGF-β1 PLUS(Qk010).TGF-β1 PLUS used at 1 ng/ml.
All experiments have been conducted by the specialist stem cell biotechnology company, Stemnovate Limited, in Cambridge, UK.
Our recombinant human TGF-β1 PLUS protein requires careful handling due to its poor solubility 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 before dilution in cell culture media or final working solutions. 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 storage.
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 0.22
Recovered concentration: 0.22 cm-1 x 10 / 2.21 cm-1 mg ml-1 = 1.00 mg / ml
Concentration was calculated using extinction coefficient at 280 nm. Example data from Qk010 batch #012.