Summary
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Bioactivity
Recombinant bovine/porcine FGF2-G3 activity is determined using the Promega serum response element luciferase reporter assay (*) in transfected HEK293T cells. EC50 = 415 pg/ml (24 pM). Cells are treated in triplicate with a serial dilution of FGF2-G3 for 3 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. Data from Qk081 lot #204612. *Promega pGL4.33[luc2P/SRE/Hygro] #E1340
Purity
Recombinant bovine/porcine FGF2-G3 154aa migrates as a major band at approximately 17 kDa (monomer) in reduced (R) and non-reduced (NR) conditions. The dimeric form is also observed at approximately 34 kDa in the non-reduced condition. No contaminating protein bands are present. The purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptoethanol, R) and non-reduced (NR) conditions and stained with Coomassie Brilliant Blue R250. Data from Qk081 lot #204612.
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Wild-type bovine/porcine FGF-2 and bovine/porcine FGF2-G3 have equivalent bioactivity
FGF2-G3 WT FGF-2
Bovine/porcine FGF2-G3 activity is determined using the Promega serum response element luciferase reporter assay (*) in transfected HEK293T cells. Cells are treated in triplicate with a serial dilution of FGF2-G3 for 3 hours. Qk081 bovine/porcine FGF2-G3 (EC50 0.209 ng/ml) and Qk056 WT FGF-2 (EC50 0.236 ng/ml). *Promega pGL4.33[luc2P/SRE/Hygro] #E1340
Compared to wild-type bovine/porcine FGF-2, thermostable bovine/porcine FGF2-G3 retains activity after pre-incubation with conditioned media at 37°C for 48 hours
Wild-type FGF-2
FGF2-G3
0 hours + 48 hours
WT FGF-2 (Qk056) and FGF2-G3 (Qk081) were diluted in conditioned media and incubated at 37 °C. Samples were taken at 0 and 48 hours. FGF-2 activity was assayed in triplicate using the Promega serum response element luciferase reporter assay (*) in transfected HEK293T cells. Results were then normalized to the maximum response for 0 hours. Comparison between Qk056 WT FGF-2 at 0 hours (EC50 0.105 ng/ml) and 48 hours (no EC50). Comparison between Qk081 FGF2-G3 at 0 hours (EC50 0.128 ng/ml) and 48 hours (EC50 0.103 ng/ml)/
Protein background
FGF-2 (also known as basic FGF or bFGF) is an essential growth factor for maintaining embryonic stem cell (ESC) and induced pluripotency stem cell (iPSC) pluripotency in feeder-free and chemically defined stem cell media. It is a core component of widely adopted media including mTESR [1], StemPRO [2] and E8 [3]. However, FGF-2 is inherently unstable and prone to proteolytic degradation and aggregation. This fundamental biochemical instability, and therefore low half-life in culture media (<10 h), is an important contribution to the need for frequent media changes and challenges in improving homogeneity during stem cell proliferation and subsequent differentiation.
Bovine/porcine FGF2-G3 can be a core ingredient in Beefy-9 media, established by Stout et al. in 2023. The innovation of adding recombinant albumin to B8 media has yielded a groundbreaking advancement in cell culture for the growth of bovine satellite cells, resulting in the development of Beefy-9 media. This novel medium is the first of its kind, meeting fundamental criteria to serve as a foundational medium for cultured meat production. One key feature includes the complete absence of animal-derived components, ensuring animal-component-free culture conditions. This departure from traditional serum-containing media, which often rely on animal-derived additives, distinguishes Beefy-9’s commitment to ethical and sustainable practices in cell culture [4-6].