With our new lineup of growth factors, we want to make sure that the products we are selling are the best possible combination of price and performance. One of our main challenges then is to make sure that each growth factor we have is able to function correctly in a specific biological assay. That may sound simple enough, but given the large number of roles that each growth factor can play and the variety of different types of factors that researchers demand, it’s not as straightforward as it may seem. For this blog post we will go through an example of the biological assay we use to test our FGF’s, a mitogenic assay of BaF3 cells to determine the ED50.
After the recombinant FGF has been produced by the E. coli cells, purified, then lyophilized, we want to make sure the protein we produced is functioning correctly and there weren’t any issues during production. First we need to have our BaF3 cells produce the necessary receptor proteins on their membranes. Certain FGF’s bind more efficiently to different FGF receptor proteins, so we make sure to test a few different FGFR’s to make sure our protein is binding correctly. The best way to control for receptor presence is to transfect the cells with an expression vector containing the necessary receptor, and select with an antibiotic specific to that plasmid. Once the cells are subcloned and are expressing the receptor proteins, we then start the proliferation assay. We aliquot cells into a 96well plate, then add a few different dilutions of either our newly produced FGF or a reference FGF. After the cells have had time to incubate with the additional FGF, we measure their mitogenic activity by adding [3H]thymidine, and after a few hours measure how much was incorporated using a scintillation counter. We are then able to tell if our cells had a significant increase in mitogenic activity after the addition of the FGF over a cell-only baseline, and how our new product compares to our reference standard.
If this data looks good, and the product passes our other quality controls (such as purity and endotoxin levels), then it is able to be sold to our customers. The biological assay will vary between growth factors, but the results are available in our certificate of analysis. Thanks for reading, and if you would like to know more about a specific product or our testing methods, please email firstname.lastname@example.org and we will try and provide you with as much information as we can.
Ornitz, D.M. et al. (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271, 15292-15297.
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