I’m seeking expert advice from anyone with experience in protein purification. My project involves depleting fibronectin (FN) from fetal bovine serum (FBS) to use the FN-depleted serum in routine cell culture experiments.
I plan to use Gelatin-Sepharose 4B resin along with Pierce centrifuge columns for the depletion process. However, I couldn’t find specific information about fibronectin levels in FBS—only the total protein concentration is listed on the product datasheet.
Has anyone performed FN depletion from FBS before?
I would appreciate guidance on the appropriate ratio of resin to serum needed to effectively deplete fibronectin.
Hello Munirah Faleh,
The exact fibronectin concentration isn't usually provided because the amount of fibronectin in FBS can fluctuate significantly. This variability makes it difficult to provide a specific, reliable concentration for every batch. The variability in fibronectin content stems from factors like the source of the serum, the processing methods, and the breed of the bovine.
If you are interested in knowing the fibronectin concentration in FBS, you may use ELISA. ELISA kits are available for quantifying fibronectin in FBS.
As far as the ratio of resin to serum is concerned, use 4ml of Gelatin-Sepharose 4B resin per 25ml of FBS. The ratio in terms of ml of resin per ml of FBS is 0.16ml resin/ml FBS. This ratio ensures efficient binding and removal of fibronectin.
As far as chemical composition of fetal bovine serum is concerned, you may want to refer to the article attached below.
https://www.researchgate.net/publication/227755611_Influence_of_diamond-like_carbon_coating_on_the_corrosion_resistance_of_the_NITINOL_shape_memory_alloy
Best,
Malcolm Nobre could I kindly ask you to provide references for the ratio of 0.16 mL of resin per mL of FBS used in fibronectin depletion, as I need to document this information in my thesis. Thank you again
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