My friend asks:
We have to block protein transport in vessel tissue culture, is it reasonable to incubate small pieces of tissue in collagenase/DNAse cocktail with Brefeldin A for about 1,5h (tissue lysis is a step of our work and we have to block protein transport at this time)? Does it work only when added, so when we wash the cells, protein transport starts again?
Waiting for your suggestions!
BFA is toxic. you do not say what type of cultured cells you are using, but even in cell types where BFA acts very fast it still takes about 20-30 mins. usually what happens is that BFA degrades the golgi apparatus and causes all the signaling compartments to aggregate (forming a large aggregate known as the "BFA body") and eventually decompose. as a first step it does block recycling, but I think there might be more specific inhibitors that block protein transport without destroying cells? at any rate, the effects of BFA are reversible provided the cells are not too far gone.
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