Proteins with high apparent pi value are often tricky because they tend to bind anionic substances such as nucleic acids, other proteins, glass, etc. Conversely given that most proteins have acidic-ish apparent pi its often worth looking into why a particular protein is basic as it may be a fact of biological relevance! Basic proteins can be purified using acidic resins which is often a boon due to the same considerations (less competition, easy separation). Of course the actual pi of a protein can deviate substantially from its theoretical pi however it is fairly safe to assume that for a theoretical pi of 9 the apprent pi will be at least above 8 provided there is sufficient *number* of charged groups (I.e. this prediction is not based on a lonely lysine and protein nterm quietly crying in its corner). The more charges there are, the more likely theoretical and apparent pi are at least in the same ballpark...
His-based affinity chromatogaphy on immobilized metal works progressively less effectively with ph decrease downwards from 7, pretty much regardless of protein pi since his tag is not often involved in local interactions (and if it is then it typically works badly as affinity tag due to steric problems anyway). Ph 4 - 5 typically would elute bound protein off imac and is in fact an optional elution metho if for some reason imidazole or histidine are undesirable. Your mileage will vary. Artem On Mar 2, 2012 6:17 AM, "Carlos Kikuti" <kik...@gmail.com> wrote: > You might be giving too much importance to the THEORETICAL pI of the > protein. If it's supposed to be well charged at pH 7,4 (only a titration > curve, and not simply knowing the pI will tell you this) and it's still > precipitating, the problem might be due to a bad fold, for instance, or to > the lack of salt ... > > I've heard people saying that proteins with high pIs are more difficult to > work with, but to be honest I don't know where that fear comes from. > > Carlos > > Em 02/03/2012, às 05:55, Artem Evdokimov escreveu: > > > This pH is generally incompatible with Ni IMAC, sorry :) If you have a > > high pI your best bet is to employ ion exchange as primary capture, > > specifically SP resin or if you're really lucky - CM resin. There are > > only relatively few proteins in E. coli that bind to CM resin at pH 5 > > and virtually none (one-three) that will bind at pH 8. If your protein > > still binds, then you're good to go. > > > > Artem > > > > On Thu, Mar 1, 2012 at 10:49 PM, anita p <crystals...@gmail.com> wrote: > >> Hi all, > >> Has anyone used sodium acetate buffer pH (4-5) for purifying histag > protein > >> on Histrap column (AKTA) followed by SEC? > >> My protein has a pI of 9. I tried pH7.4 but it has precipitation > problems. > >> While doing buffer screening using 24 well hanging drop I found that > lower > >> pI onces are clear, so just thinking can I use Na acetate at pH 5 for > whole > >> purification??? > >> > >> > >> Thanks in advance > >> Anita >
