Fibronectin coating: BSA vs no BSA - (Sep/26/2010 )
This is going to be the very first time I coat my tissue culture plate with fibronectin. I found several protocols online but a special one from Millipore made me a little confused.
The Millipore's protocol mentions that after fibronectin coating, BSA blocking is necessary (http://www.millipore...ech1/mcproto007). Can anyone explain to me the rationale of blocking the coated plate with BSA before growing cells on them? I thought the cells require fibronectin for adhering and proliferating????
The other protocols I found just mentioned removing the fibronectin solution, washing the coated surface with Mg2+, Ca2+ free PBS once, and growing the cells on the coated plate.
Another question is, what is the stability of fibronectin? The fibronectin solution from Sigma in my lab has been sitting in the fridge unopen for ages, at least 4 years.
Also, in some protocols, it is mentioned that fibronectin can not be freeze-thaw repeatedly. How do you normally handle it? Do you dilute it to your working concentration and freeze it in small aliquots?
Many thanks in advance.
The protocol from Millipore is probably talking about coating a plate with fibronectin for an adhesion assay (the link is broken). For an adhesion assay, you usually coat overnight with fibronectin, wash the plate and block with BSA to prevent non-specific interactions (as you want the cells to bind to fibronectin). Whereas, you want to coat your plate to grow cells on, so you don't need to block with BSA.
Our fibronectin solution has been in the fridge for a while and still works. Our fibronectin solution (sigma)says that it must be stored at 4-8 degrees. I dilute mine fresh each time I make it.
Hope that helps..