What is the actual process that leads to fibronectin coating glass dishes?
As far as I understand for collagen it works, by bringing the solution's ph to the isoelectric point to make it insoluble, for laminin which is stored at -24 to -80 degrees it gelates at room temperature.
But fibronectin's isoelectric point is around 5.5-6, it is stored in water at 4 degrees and usually diluted in PBS (PH ~7.4) while I heard of people coating dishes in with fibronectin in the fridge as well.
So neither of the two processes explain why the fibronectin is attaching to the glass plate.
Can anyone explain why fibronectin is coating the well plates?
Hi Max Heydasch . Protein binding to tissue culture plastic and ELISA plates are mostly hydrophobic in nature. For silica, it is mostly electrostatic.
The surface chemistry is very important since adhesion proteins dont readily bind bacterial plastic plates and treating glass with hydrophobic silanes can negate protein binding.
Hi Max. I'm only offering observations, which may provide additional clues to the answer you seek.
We routinely coat glass and TC plastic with fibronectin in PBS pH7.4, overnight at 4oC. In a hurry, this can be done at r.t. or 37oC, 1 hr. We can also get BSA to coat with the same process, and a bunch of other non-ECM proteins as well (recombinant GST-proteins for example). I always assumed its electrostatic. TC plastic coats more readily than glass coverslips, in my experience. Our slips are not acid-washed prior to coating; not necessary at all. But its possible that quality coverslips came pre-washed. Once, I kept coverslips in 70% ethanol to have a sterile batch that is ready to use. Bad idea, those never coated, despite having rinsed off the ethanol. I thought that was strange.
At low concentrations, Fibronectin appears to coat weakly as a film of sorts. That is because harsh handling (such as PBS rinse) can strip the coating quite easily. This would be easily noticeable as a non-wetting area of the glass, or hydrophobic-like pooling of water or PBS.
Finally, PDMS silicone is hydrophobic and near impossible to coat. While some folks use cold plasma to activate/charge the surface, we found that good quality Fn at higher concentrations can be sufficient to coat PDMS.
hope that helps.
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