Use cold TCA for protein precipitation? - (Jan/25/2008 )
Hello everybody,
It seems an old topic but many details keep unanswered on this forum, to the best of my knowledge.
It seems that to precipitate proteins using TCA (trichloroacetic acid) from cell lysate, some use TCA in room temperature while others put TCA on ice before use. Would this really make a difference?
What I can think of is a better protection of things of interest by this low temperature. But is TCA really chemically reactive and so is a concern?
Disadvantage using cold TCA could be that precipitation efficiency is lower at lower temperature because slower Brownian motion of protein molecules means less chance of collision between these molecules and thus slower formation of protein clumps (clumps more readily precipitate).
So if using cold TCA, one has to wait for a longer time (even longer if TCA-cell lysate mixture is put in fridge, not on benchtop). How long is long enough?
Thanks
It seems an old topic but many details keep unanswered on this forum, to the best of my knowledge.
It seems that to precipitate proteins using TCA (trichloroacetic acid) from cell lysate, some use TCA in room temperature while others put TCA on ice before use. Would this really make a difference?
What I can think of is a better protection of things of interest by this low temperature. But is TCA really chemically reactive and so is a concern?
Disadvantage using cold TCA could be that precipitation efficiency is lower at lower temperature because slower Brownian motion of protein molecules means less chance of collision between these molecules and thus slower formation of protein clumps (clumps more readily precipitate).
So if using cold TCA, one has to wait for a longer time (even longer if TCA-cell lysate mixture is put in fridge, not on benchtop). How long is long enough?
Thanks
denaturation of proteins makes them cleavable for proteases which are always abundant in cell lysates; cooling TCA minimizes protease activity which may have activity even under harsh conditions...
moreover tca should be kept at 4° to minimize fumes when opening the bottle as it's high corrosive.
protein precipitation, including by tca, is more complete when performed at low temperatures. not just because of a protective effect but also because everything, except gasses, are less soluble at low temperatures.
so, if you are reducing solubility of the protein with the addition of salt or tca or peg or..., then you will get a more effective precipitation at low temperature.
with tca, however, you are also denaturing the protein.