Qk002 Recombinant zebrafish FGF2 protein (bFGF) is a 17 kDa biologically active domain of zebrafish fibroblast growth factor 2 protein, which has been optimized for expression in E. coli and purified to homogeneity. Our recombinant FGF2 protein is trusted by the Cambridge Stem Cell Institute, UK to support the maintenance and differentiation of their stem cell cultures.
Recombinant zebrafish FGF2 protein
£250.00 – £400.00
Qk002 Recombinant zebrafish FGF2 protein
Fibroblast growth factor 2 (FGF2) – also known as basic fibroblast growth factor (bFGF) – has a broad range of physiological roles including regulation of cell proliferation and differentiation. FGF2 is used to support the maintenance of human embryonic stem cells and proliferation and differentiation of induced pluripotent and mesenchymal stem cells.
Recombinant zebrafish fibroblast growth factor 2 has been used extensively to support the maintenance of human stem cells (1–3). This version of recombinant zebrafish FGF2 is used by the core facility at the Cambridge Stem Cell Institute, UK and historically by many of the stem cell groups based at University of Cambridge.
Summary: Mature domain of zebrafish Danio rerio FGF2/bFGF (residues 2-154, Uniprot: B3DGE3) expressed in E.coli and purified to homogeneity as described in Ludwig et al. (2).
Form: Protein is provided in PBS without carrier protein at 2 mg/ml.
Molecular mass: ~17 kDa
Basic fibroblast growth factor, bFGF, FGFβ, FGF2, Fibroblast growth factor-basic, HBGF2
1. Ludwig, T. E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).
2. Ludwig, T. E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).
3. Chen, G., Gulbranson, D. R., Yu, P., Hou, Z. & Thomson, J. A. Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells 30, 623–30 (2012).
Purity and bioactivity
For peace of mind that our recombinant zebrafish FGF2 protein (also known as bFGF) 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 zebrafish FGF2 protein and ensure that its purity meets are rigorous standards, we conduct SDS-PAGE on every protein batch in both reduced and non-reduced conditions and use appropriate loading and high sensitivity stains.
We confirm consistent bioactivity using a FGF-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 for each preparation of our recombinant zebrafish FGF2 protein.
Find out more about our extensive purity testing in the next tab.
Protein purity: SDS-PAGE in reduced and non-reduced conditions
Bioactivity: FGF-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: Result: FGF2 migrates as major band at 17 kDa in non-reducing (-βME) conditions and upon reduction (+βME). The higher molecular mass band at 35 kDa is a dimer that we always see in our highly purified zebrafish FGF2 protein, the presence of this does not affect biological activity.
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.
Bioactivity: luciferase reporter assay
Purity: mass spec analysis
Result: calculated molecular mass of FGF2 is 17036 Da. Molecular mass from this analysis is 17035 Da, consistent with the calculated mass. The calculated molecular mass of dimeric protein is 34064 Da and this is represented in the mass spec with average molecular mass 34062 Da.
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.
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.
Thaw the sample on ice, spin briefly and dilute with PBS as needed. Our protein are supplied carrier-protein free. If compatible with your work, add a carrier protein of your choice such as BSA, HSA or gelatin to further minimize loss by adsorption. Spin in a microfuge for 5 minutes at maximum speed and divide the solution into suitable aliquots and store at -80°C. We recommend that single-use aliquots should be prepared to avoid freeze-thaw cycles.
Every effort is made to ensure samples are sterile; however, we recommend sterile filtering after dilution in media or the final working solution.
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 that 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 0.093
Recovered concentration: 0.093 cm-1 x 20 / 0.935 cm-1 mg ml-1 = 1.99 mg / ml
The sample was diluted 1:10 in 100 mM sodium phosphate pH 7.4 and the UV spectrum 340-220 nm measured in duplicate. Concentration was calculated using extinction coefficient at 280 nm.