His tag protein purification problem - (Jun/12/2004 )
Thankyou for all your suggestions.
As a final option, i scanned for different NaCl concentrations at which the Protein can show binding to Ni NTA.
The Ni NTA to His interaction is so specific that it remains at such high concentrations of NaCl like 1M or even 2M.
The protein that initially creared problem in not binding to any column, bound very well to Ni NTA itself at 1M NaCl in buffer.
I suggest sincerely, if someone has problem in Ni NTA (His tag binding problem) to try higher salt concentrations before concluding any thing.
Hi All
I have very less experience with His tagged protein purification. After purifying my protein (30kDa) using Ni2+ resin I still see other proteins in the eluted fraction. One of my colegue demands that by re-purifying those fractions in the Ni2+ resin once again under exactly the same conditions he can clean it up . Is that possible?
Thanks for your suggestions,
Somali
---Yes, Its including the Tag
did you pH the buffers very carefully? do not follow Maniatis' protocols of mixing powders at some ratios, as the resulting pH can easily be 0.2-0.5 different and it DOES make quite a difference in affinity work.
did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!
Yes, i dont follow the mixing powders...i adjusted pH by HCl Concentrated for Tris. to pH 8.0
did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!
Yes i did...no use..
Did you have DTT in all your solutions your protein was in? This includes lysis buffers, dialysis buffers etc. 1mM DTT will prevent the majority of missfolding events.
No i didnt ..will try defenitely
Your first objective would be to track the protein size using a Sephacryl S-500 gel filtration and check if the target entity exists as a dimer/trimer/teramer or multiples thereof. Inclusive tight aggregates prevent binding of proteins to columns. RNA polymerase core enzyme comprising of alpha and the beta/beta-prime subunits with a piece of embedded DNA does not bind to any column despite presence of denaturants. Could your target entity be a DNA or RNA binding protein?
Yes..i tried putting on Sephadex G75 and Sephadex G200 from Amersham on Akta FPLC..all the cases the protein came in void volume.
Its supposed to be a DNA cutting and rejoining enzyme (integrase) but its site specific.
Hi,
Since you tried Gel filtration, and your protein always came in void volume. This indicates your protein may be aggregated, right?
Guodong
I have very less experience with His tagged protein purification. After purifying my protein (30kDa) using Ni2+ resin I still see other proteins in the eluted fraction. One of my colegue demands that by re-purifying those fractions in the Ni2+ resin once again under exactly the same conditions he can clean it up . Is that possible?
Thanks for your suggestions,
Somali
rechromatography on the previously used column under same conditions does help sometime (not always). the binding and elution profile of a protein preparation depends not only on the nature of the protein but also on the relative abundance of other proteins. When the protein concentration is too high in the first run, the elution profile is not good. in the second run, the number of other proeins are less and the elution profile is imporoved.
there has been instances in our department where people have used octyl sepahrose chromatography succesviley with improved resolution on the rechromatogram. one protein was purified by ion-exchange before and after a gel filtration step.
sometimes, chagning the sequence of the columns also helps. i used phenyl sepharose as the first step for a certain protein and thereafter a gel filtration; always, there was another band who couldn't be removed by manipulating these chromatographies. I included an ion exchange run ( Q-sepharose) before phenyl and got good purification.
Sharath Balakrishna
Research Fellow
National Chemical Laboraotry,
India
Since you tried Gel filtration, and your protein always came in void volume. This indicates your protein may be aggregated, right?
Guodong
Hi,
Initially i thought so, but, after that i had a doubt that it could be ionic interaction. So, i tried higher and higher NaCl concentration in buffer (1M). Which helped.
Afer elution i could buffer exchange the protein in low salt buffer (150 mM) without any problem by dialysis.
sankar
Did you notice the flow rate? If the flow is very fast the protein may not combine with the resin.So we always control the flow rate in quite slow (perhaps 10ml/40min) when we add the lysis buffer to the resin,and to completely bind the protein to the Ni column we offten add them twice. 
Hi all
I am also having problems with Ni NTA purification. I am working with an rna binding protein which is a 128Kda protein with theoretical PI of 7.1. With a thioredoxin tag at the N-terminal, and v5 and 6X his at the c terminal the final MW is 144Kda and PI is 6.28. I could express it with pBAD promotor.
Its positive in the western. But I am having problems in purifying it. It is not binding to Ni NTA column. I tried someof the suggestions listed in this discussion, but not yet successful.
Any specific suggestions are very much appreciated
Thanks and regards
Srinivas
Sorry for asking this silly question. What is thioredioxin? what is its advantage?
I am trying to Ni-NTA purify scFv antibody fragements. Although I get good expression & yield of periplasmic protein, mojority of proteins eluted are infact ecoli proteins (I have sequenced them!) with my protein representing less than 1% of the eluted protein. Can you suggest clean up procedures (Ion exchange?), dose anyone else see this? Cheers
Hi,
I faced the same situation a few months back. Expexted size of my proten was 43 kd. there was clear induction. Intially there was solubilty problem which i solved with lowering the iptg level.The protein was picked up in the western but it never bound to Ni-NTA bead.I thought the His tag is overwhelmed by the protein, then i did the denatured purification of the protein still the protein could not bind to the bead.
To my surprise it was a heat shock protein getting oveexpressed. you change the expression cell. This will give you the clear picture.
Ashraf