Recombinant human FGF-4 protein (Qk004)2020-03-30T14:43:10+00:00

Recombinant human FGF4 protein (Qk004)

Fibroblast growth factor 4 (FGF-4) is a member of the FGF superfamily with a physiological role in the regulation of proliferation and differentiation in embryonic stem cells and tissue stem cells (1–3)

FGF-4 promotes neural stem cell proliferation and neuronal differentiation in the postnatal brain (4), increase the proliferation rate of human adult bone-marrow derived mesenchymal stem cells (5) and support the maintenance, proliferation and self-renewal properties of human embryonic stem cells (6).

Synergism between FGF-4 and WNT signalling acts to form hindgut organoids from iPSC-derived human posterior gut endoderm cells (7) and FGF-4 is used to mimic embryonic intestinal development during directed differentiation in culture of pluripotent stem cells into intestinal organoids.

Summary: Mature domain of human FGF4 (residues 79-206, Uniprot:P08620) expressed in E.coli and purified to homogeneity. Mature protein is a non-glycosylated protein.

Molecular mass: ~14 kDa

Form: protein is provided frozen in PBS (carrier protein-free) at 1 mg/ml

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 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.

  1. Beenken, A. & Mohammadi, M. The FGF family: biology, pathophysiology and therapy. Nat. Rev. Drug Discov. 8, 235–53 (2009).
  2. Coutu, D. L. & Galipeau, J. Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging (Albany. NY). 3, 920–33 (2011).
  3. Kosaka, N., Sakamoto, H., Terada, M. & Ochiya, T. Pleiotropic function of FGF-4: Its role in development and stem cells. Dev. Dyn. 238, 265–276 (2009).
  4. Kosaka, N. et al. FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation. FASEB J. 20, 1484–1485 (2006).
  5. Farré, J. et al. FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells. Growth Factors 25, 71–76 (2007).
  6. Mayshar, Y. et al. Fibroblast Growth Factor 4 and Its Novel Splice Isoform Have Opposing Effects on the Maintenance of Human Embryonic Stem Cell Self-Renewal. Stem Cells 26, 767–774 (2008).
  7. Zhang, R.-R. et al. Stem Cell Reports Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids. Stem Cell Reports 10, 780–793 (2018).
  8. McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).
Fibroblast Growth Factor-4, Kaposi’s sarcoma-associated FGF, k-FGF, Heparin secretory-transforming protein 1 HST-1, Transforming protein KS3, Heparin-binding growth factor 4, HBGF-4

Result: FGF4 migrates as a single band at 14 kDa in non-reducing (NR) and upon reduction (R).  No contaminating protein bands are visible.

Recombinant FGF-4 protein purity in SDS-PAGE

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.

Result: FGF4 induces cell proliferation of HEK293T cells.

Human embryonic kidney cell line 293T (HEK293T) was cultured in serum-free media containing 100ng/ml hFGF2. The number of viable metabolically active cells was detected by ATP measurement using a bioluminescent reaction (n=3; data show mean ± SEM. Data provided by Stemnovate Ltd, Cambridge, UK.

Result: FGF4 induces cell proliferation of iPSC cells (Stemnovate iPSC1 line).

IPSC (Stemnovate iPSC1) were cultured in serum-free media containing 100ng/ml hFGF4. The number of viable metabolically active cells was detected by ATP measurement using a bioluminescent reaction (n=3; data show mean ± SEM). No media change was performed on day 2 leading to reduced viable cell numbers on day 3. Data provided by Stemnovate Ltd, Cambridge, UK.

Result: calculated molecular mass of the FGF4 is 14409.8 Da. Result of the analysis: 14278.4 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.

Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.

Absorbance at 280 nm: average 0.068
Recovered concentration: 0.068 cm-1 x 10 / 0.62 cm-1 mg ml-1 = 1.1 mg / ml
Recovery: 110% (>100% due to routine 10% over-fill of vials during aliquoting)

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

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.

View full batch quality testing data for Qk001

Batch #010

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.

When we test our proteins, we choose a vial at random and reconstitute as recommended to ensure we are testing as close to the protein you will receive as possible.  Biochemical identity and purity is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography.  Bioactivity is determined 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.

Please contact us with questions any time by email 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, whichever is easiest for you, or email your PO to orders@qkine.com.

We also 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.