This is a very general question, I know, but I've been purifying proteins and exploring expression/purification conditions in E.coli during the last couple of months and I'm having trouble to find the explanation for the use of some molecules in some purification protocols.
The addition of ATP/MgCl2 and lower salt concentrations during some protocols for protein purification is an example of that. I understood that the addition of these components can help getting rid of some contaminants, namely chaperons, but I never got a real explanation of how it works at the molecular level.
Many chaperones use ATP hydrolysis as the energy source for completing their binding-renaturing-release cycle (the hsp70 family, for instance), so if a semipurified protein contains chaperone/protein complexes, adding ATP enables the chaperone to complete the cycle and detach from the substrate (enabling further purification of the target protein). Mg++, I am guessing, is added only to stabilize the triphosphate moiety.
Many chaperones use ATP hydrolysis as the energy source for completing their binding-renaturing-release cycle (the hsp70 family, for instance), so if a semipurified protein contains chaperone/protein complexes, adding ATP enables the chaperone to complete the cycle and detach from the substrate (enabling further purification of the target protein). Mg++, I am guessing, is added only to stabilize the triphosphate moiety.
As you can see have been working with the muscle tissues? In this case addition of MgATP (NaATP plus Mg2Cl added at equal molar concentration; do not buy Mg ATP it is very expensive and is exactly the same) this is absolutely necessary for muscle relaxation. Perhaps in the case of haperons the situation is similar but most likely as in the muscle you have to remove CaCl2 by addition of 2 mM of EGTA. Actually in smooth muscle one have to remove both Ca++ and Mg++ with 2-5 mM EDTA. More important is what kind of proteins you are trying to extract; and what do you need them for? Do you see them on SDS PAGE of your extracts?
For a simple extraction I would suggest to use a solution at pH about 10, buffered with 10 mM EDTA. You can be sure to extract everything by adding to this buffer 8 M urea but this will denaturate your proteins. They may renaturate if rather small.
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