From: anita p <crystals.rp
Date: 2 March 2012 04:49
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
----------
From: Artem Evdokimov
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
----------
From: Carlos Kikuti
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:
----------
From: Artem Evdokimov
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
----------
From: Roger Rowlett
----------
From: Santosh
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/
----------
From: Kevin Jin
Date: 2 March 2012 04:49
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
----------
From: Artem Evdokimov
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
----------
From: Carlos Kikuti
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:
----------
From: Artem Evdokimov
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
----------
From: Roger Rowlett
Classical protein purification by IEX, HIC, GEC, etc. is apparently a dying art. It is typically quite easy to purify proteins using non-affinity methods from overexpression mixtures using an AKTA system. (Gosh, in the old days we used to purify to homogeneity proteins with 0.1% abundance in a natural source--a 15% overexpression crude extract is trivial by comparison.) It is fairly quick to scout good step gradient conditions for partially purifying your protein by ion exchange by using a 1 mL or 5 mL ion-exchange column, then scale up to a 1.6x10 cm or 2.6x10 cm column, depending on your crude extract sample volume. (We maintain Q- and SP-sepharose columns in our lab for low- and high-pI proteins, respectively.) A secondary purification via hydrophobic interaction or even salt fractionation is typically sufficient to clean up well-overexpressed proteins. Desalting and polishing can be accomplished on a large (1.6x60 cm) gel exclusion column. We purify our current crop of proteins we are studying this way--no tags to remove later. Sometimes, the time saved by affinity purification on the front end is eaten up on the back end with the necessity for purification tag removal. Don't be afraid to give the "old" methods a go. As Artem has pointed out, if your target protein has an unusual pI, it may essentially purify in one step (IEX) and you can then do a quick polish on GEC.
Cheers,
_______________________________________
Roger S. Rowlett
Cheers,
_______________________________________
Roger S. Rowlett
----------
From: Santosh
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/
----------
From: Kevin Jin
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
From: Paul Kraft
Date: 2 March 2012 17:03
I have purified dozens of very high pI viral proteins, and I can't stress enough the requirement to wash your protein with at least 1M NaCl after binding it to the histidine collumn. DNA fragments often require a 2M NaCl wash. High pI proteins are very soluble.
Dr. Paul Kraft
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