Maybe you can this way. Use Na acetate to elute your protein, then use EDTA to remove the Ni from your protein, then buffer exchange or dialysis to remove EDTA.
Kevin On Fri, Mar 2, 2012 at 5:50 AM, Santosh <shodawade...@gmail.com> wrote: > Hi Anita, > As Artem noted, use of Histrap column at lower pH would not be a great idea > and unless you want to elute your protein using pH Step gradient > http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/A960DBAAE1C0C945C1257628001D29BB/$file/28404480AA.pdf > You may also consider using CM (Carboxy Methyl weak cation exchanger) column > at pH6.8 and load the peak fractions of your protein directly on Histrap in > 20mM Hepes pH6.8 in higher salt ( match the conductivity using conductivity > meter, high salt will rescue your protein from falling out of solution to > some extent, you can always optimize salt up to 500mM) > At this point you can concentrate your peak fractions in buffer of your > choice before loading on SEC using your buffer of choice. Check for > aggregates on peak fractions using DLS you may also be able to screen > buffers using DLS that has 384 well plate set up. It is important to know > the range of different buffers and their long term shelf life and stability. > http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html > I hope it helps you figure out the optimum pH conditions for you target > protein. > Best, > Santosh Hodawadekar, PhD > http://www.linkedin.com/in/shodawadekar/ > > > On Fri, Mar 2, 2012 at 8:14 AM, Artem Evdokimov <artem.evdoki...@gmail.com> > wrote: >> >> 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 > >
