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Protein doesn't bind any column except Ni


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#1 vxmqzz

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Posted 13 March 2009 - 12:53 PM

I am purifying a c-terminal his tagged protein. It expresses well and binds to Ni column and elutes out around 250mM Imidazole. When I try to further purify it, it won't bind MonoQ or MonoS column at varies PH, always comes out in flow through. During Gel filtration it comes out in void volume. I suspect the protein somehow aggregates but remains soluble. Is there anyway I can purify it without denaturing it ?

Thanks

#2 klinmed

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Posted 13 March 2009 - 01:26 PM

I am purifying a c-terminal his tagged protein. It expresses well and binds to Ni column and elutes out around 250mM Imidazole. When I try to further purify it, it won't bind MonoQ or MonoS column at varies PH, always comes out in flow through. During Gel filtration it comes out in void volume. I suspect the protein somehow aggregates but remains soluble. Is there anyway I can purify it without denaturing it ?

Thanks


I find it very hard to believe that your protein does not bind to either MonoQ or MonoS columns. At an appropriate pH (and low ionic strength), all proteins bind to either an anion or cation exchangers I firstly assume that you are attempting to bind in a low ionic stength (ca 25 mM) buffer suitable for the exchanger being used (eg Tris for Q and acetate for S). Also that your sample has been desalted into the ion-exchange start buffer. Have you tried binding in 25 mM Tris-Cl pH 9.5 with monoQ or pH 3.5 with monoS? These may seem like extremes but they are good starting points. I would be very surprised if your material fails to bind under one of these conditions.

As for the gel permeation result, it sounds as if your protein may be forming soluble aggregates. Have you tried running the column in the presence of imdazole (his tagged proteins sometime aggregate?.)
How does your protein look on a native PAGE and IEF gels?

#3 T C

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Posted 13 March 2009 - 08:02 PM

Hey

Check the isoelectric point of your protein. The buffer pH should be such that yr protein is appropriately charged for the matrix (-ve for anion exchange and +ve for cation exchanger). So if Pi is say 4 and I want to do anion excahnge, I would use a buffer with pH of more than 4 (say 100 mM phosphate pH 7.2)

Hope it helps.

TC

#4 vxmqzz

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Posted 13 March 2009 - 10:38 PM

I am purifying a c-terminal his tagged protein. It expresses well and binds to Ni column and elutes out around 250mM Imidazole. When I try to further purify it, it won't bind MonoQ or MonoS column at varies PH, always comes out in flow through. During Gel filtration it comes out in void volume. I suspect the protein somehow aggregates but remains soluble. Is there anyway I can purify it without denaturing it ?

Thanks


I find it very hard to believe that your protein does not bind to either MonoQ or MonoS columns. At an appropriate pH (and low ionic strength), all proteins bind to either an anion or cation exchangers I firstly assume that you are attempting to bind in a low ionic stength (ca 25 mM) buffer suitable for the exchanger being used (eg Tris for Q and acetate for S). Also that your sample has been desalted into the ion-exchange start buffer. Have you tried binding in 25 mM Tris-Cl pH 9.5 with monoQ or pH 3.5 with monoS? These may seem like extremes but they are good starting points. I would be very surprised if your material fails to bind under one of these conditions.

As for the gel permeation result, it sounds as if your protein may be forming soluble aggregates. Have you tried running the column in the presence of imdazole (his tagged proteins sometime aggregate?.)
How does your protein look on a native PAGE and IEF gels?



Thanks for the reply. I tried PH 8.5 for MonoQ and PH 5.5 for MonoS and I did get salt concertation to very low levels, I might need to try extreme PHs as you suggested. The gel filtration buffer has no Imidazole and I haven't run native PAGE or IEF at the moment.

#5 klinmed

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Posted 13 March 2009 - 10:52 PM

Hey

Check the isoelectric point of your protein. The buffer pH should be such that yr protein is appropriately charged for the matrix (-ve for anion exchange and +ve for cation exchanger). So if Pi is say 4 and I want to do anion excahnge, I would use a buffer with pH of more than 4 (say 100 mM phosphate pH 7.2)

Hope it helps.

TC


Good idea to check the Pi. This can be done using IEF gels or predicted from the deduced protein sequence using Expasy tools (www.expasy.ch/tools/).

The use of a phosphate buffer with an anion exchanger is totally inappropriate. It will cause instabilities in pH, zone broadening etc during salt elution significantly lowering resolution. Suggest you have a look in the handbook written by the gel manufacturer (GE Healthcare Life Sciences) entitled "Ion-exchange chromatography and chromatofocusing: Principles and methods". It is free on their website and includes tables of appropriated buffers for the individual exchangers.

Just a thought: Seeing that this protein seems to be a bit unusual with respect to itīs binding to ion-exchangers have you tried purification by negative selection. Pass your protein through Q at 8.5 and collect flow-through, adjust to buffer for cation exchange and pass through S at <5.5 and again collect flow-through. This is an old, and neglected, technique but often gives very good degrees of purification.

Edited by klinmed, 13 March 2009 - 11:37 PM.


#6 T C

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Posted 14 March 2009 - 02:02 AM

Hey

I really don't know what the theory says but for the last five years I have been using 100mM phosphate buffer pH 7.2 for anion exchange chromatography and it works very well.

Best
TC

Hey

Check the isoelectric point of your protein. The buffer pH should be such that yr protein is appropriately charged for the matrix (-ve for anion exchange and +ve for cation exchanger). So if Pi is say 4 and I want to do anion excahnge, I would use a buffer with pH of more than 4 (say 100 mM phosphate pH 7.2)

Hope it helps.

TC


Good idea to check the Pi. This can be done using IEF gels or predicted from the deduced protein sequence using Expasy tools (www.expasy.ch/tools/).

The use of a phosphate buffer with an anion exchanger is totally inappropriate. It will cause instabilities in pH, zone broadening etc during salt elution significantly lowering resolution. Suggest you have a look in the handbook written by the gel manufacturer (GE Healthcare Life Sciences) entitled "Ion-exchange chromatography and chromatofocusing: Principles and methods". It is free on their website and includes tables of appropriated buffers for the individual exchangers.

Just a thought: Seeing that this protein seems to be a bit unusual with respect to itīs binding to ion-exchangers have you tried purification by negative selection. Pass your protein through Q at 8.5 and collect flow-through, adjust to buffer for cation exchange and pass through S at <5.5 and again collect flow-through. This is an old, and neglected, technique but often gives very good degrees of purification.



#7 swanny

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Posted 15 March 2009 - 02:46 PM

Don't make the mistake of assuming the theoretical pI is the actual pI of the folded protein, or the surface of the protein. For my PhD, I had to purify some zinc finger constructs that were virtually identical, but which bound in totally opposite pH / bead combinations. Did you try MonoQ at 5.5, or MonoS at 8.5?

I saw this protocol a few years ago, source lost unfortunately... It's basically a pH scouting technique carried out in batch mode.

Aliquot some loose anionic media in solutions of varying pH, and do the same for cationic beads. Choose a range that works well for the beads as appropriate in 0.5 pH units steps. Equilibrate the beads with a couple of washes.

Add some of your protein, adjusting the pH if necessary to maintain required pH. Incubate for 15-20 minutes, then spin down beads GENTLY (max 1000 xg ). Remove S/n and resuspend beads in buffer. Run a gel with both S/n and bead suspension. You should find a pH where the beads bind. Select a pH about 1 unit past the point that most of your protein binds to the beads.

Using that pH for your binding, repeat the binding step. split the beads into 5 or 6 fractions, then add salt at increasing concentrations; this will give you the basic elution conditions.
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#8 mikew

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Posted 17 March 2009 - 03:35 PM

After you elute off the Ni column with Imidazole do you dialyze?
If not, this may be your problem.
Dialyze to reduce the salt and get rid of your Imidazole.

#9 sudhakar mutyala

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Posted 24 April 2009 - 02:49 AM

Hi

Imidazole need to be dialyzed before going for IEX and you need to know your exact pI of your protein. Try to play with salt and pH for IEX , i Dont think there is something wrong in the GFC i presume you did not choose better resolving beads for GFC, try to get fractionation range of ur gel and use it properly.


All the best
Regards
Sudhakar

I am purifying a c-terminal his tagged protein. It expresses well and binds to Ni column and elutes out around 250mM Imidazole. When I try to further purify it, it won't bind MonoQ or MonoS column at varies PH, always comes out in flow through. During Gel filtration it comes out in void volume. I suspect the protein somehow aggregates but remains soluble. Is there anyway I can purify it without denaturing it ?

Thanks



#10 LifeTein Peptide

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Posted 07 May 2009 - 08:44 AM

That was what I am going to say. You need to dialyze the medium after Ni purification.
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#11 T C

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Posted 07 May 2009 - 08:58 AM

Hey,

I regularly use resource Q on FPLC and the loaded protein always has imidazole. In fact I plan to hook up an anion exchange column in series with the Ni-NTA column and do both the purifications together to save time.

It works.

Best,

TC

That was what I am going to say. You need to dialyze the medium after Ni purification.






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