TCA vs. meth/chloro - concentration of proteins (May/12/2006 )
hi all,
i have some problems concentrating and changing the buffer of my dilute protein solution using amicon filter units so i decided to use another protocol.
i was wondering if someone have experiences with precipitation (and at least purification) with tca or methanol/chloroform. did someone compare these two methods concerning the recovery or purity?
my protein got a his-tag, is about 60 kda and isolated under denaturated conditions by ni-nta. elution buffer contains NaH2PO4, Tris-HCl, NaCl, Urea, Imidazole. i have about 3 ml protein solution and want to concentrate the solution and change the buffer. i found several topics with similar problems and it seems that tca precipitation makes the pellets difficult to resolve. is this right?? 
so my question is: are these methods suitable for concentrating and buffer exchange of my protein solution and are there any practicable protocols without the need for high technical equipments 
??
thanks in advance for your suggestions!!
flausch
well, despite the fact the pellet is quite difficult to resuspend, it's was not a nightmare when i procceded with TCA acetone method.
But you wrote that your protein is in a buffer which contains urea. Does it necessary for protein suspesion? I was wondering if you change buffer would not your protein become insolube??...
But you wrote that your protein is in a buffer which contains urea. Does it necessary for protein suspesion? I was wondering if you change buffer would not your protein become insolube??...
hi fred,
(looks like my big fat red cat at home which snuffs the whole day too
)thanks for your answer. this is the first time i produced recombinant protein so i used a standard protocol which implicates urea in all buffers. i haven't evaluate if it is necessary for the solubility of my protein. you may wright i have to check this. but let's assume it is necessary what is the quintessence? is it not possible to change the buffer for example to pbs or any other buffer with stabilisator?
flausch
well urea helps to solubilise the proteins. So best advice is to precipitate an aliquot an tedst your second buffer on it and see what happens...