When trying to tune the stiffness of fibrin gel (50-10mg/mL) I am getting opaque gels in the 20 and 10mg/mL range. Can I get rid of it somehow?
For 100uL of 50mg/mL of gel I use 50 uL of 100mg/mL fibrinogen in PBS, 25uL of 40mM CaCl, and 20 uL of PBS. For other densities of gels 20 and 10uL of 100mg/mL fibrinogen and 50 and 60ul of PBS respectively is used
This mixture is kept on ice and polymerized by addition of 5uL of thrombin (100TIU/mL) by placing in 37C for 15 min.
Thanks for help
Hi, Vladimir.
Try spinning down all the solutions including fibrin, trhrombin and buffer, etc. at 10,000rpm for 10 min. Use thin-wall PCR tubes. I suspect that the fibrinogen and thrombin solutions, buffer, Ca and other ddH2O for dissolution may contain particles. Make all fresh solutions with autoclaved ddH2O. Get rid of particles and microscopic strings in eppen-tubes using an air-blower. Unwanted dust,strings and particles stimulates aggregated form of gel, but not even gelling at molecular level.
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