If the protein is denatured, with urea,6M, and still does not bind, then you have a problem with many of the things described by everyone. This is easy to try. Sometimes the proteins just don't bind in their native state. So you can put the protein in urea with a reding agent( even 5mM DTT is ok), and then wash, and then slowly reduce the urea concentration, to about 1M, and then wash with 1 column volume of no urea buffer, and then elute. It shou ld work as your protein was soluble to begin with. But you can check first just to see if it binds in 6 M urea by adding it to some resin. If it does, then you can proceed with the washing and refolding on the column. Believe it or not, proteins with his tags, in their native state, don't always bind to the resin. This would be easier and faster to try than making a new construct that may or may not work.

Some proteins when they fold may not have the 6xHis exposed but tucked away not allowing binding to the Ni column. you could try a mild detergent or increasing the ionic strength some. Alternatively you may have to try repositioning the tag on the N-terminus.

Hi Kevin,

I am not using detergent for my study. Also, my protein used to bind to the column before. It suddenly stopped binding. Initially I used TBS instead of PBS. Will that make a difference?

If it binded with TBS, why did you change?

Do you need the protein under native conditions? If not, Kevin's suggestions may help you. Even so, try to increase the NaCl concentration up to 500mM, or even 1M if it still doesn't work.

If you're re-using a column, there's a chance it should be recharged with new Ni ions. We use g-bioscience's resin and this is the protocol we use (end of post).

If you do not need native protein, you can try a denaturing protocol and attempt to re-fold on the column (his-tag will still associate if the protein is denatured, then wash with decreasing amounts of your denaturing agent to allow gradual refolding, does not work for all proteins though, see protocol here http://openwetware.org/wiki/Knight:Purification_of_His-tagged_proteins/Denaturing_with_refolding)

COLUMN RECHARGING PROTOCOL:

Column regeneration should be performed when a different protein is being isolated or

when there is a significant loss in the yield of protein. If the Nickel Chelating Resin loses

its blue color the column needs recharging.

1. Wash the resin with 5 column volumes of a solution 20mM sodium phosphate

supplemented with 0.5M NaCl, 50mM EDTA at pH7.0.

2. Wash with 5 column volumes of distilled water to remove EDTA.

NOTE: If the loss in yield is suspected to be due to denatured proteins or lipids a

more drastic regeneration protocol should be followed. After step 2:

A. Elimination of ionic interactions: Wash in batch for approximately 20minutes

in a solution with 1.5M NaCl,follow with a wash with 10 column volumes of

distilled water.

B. Elimination of precipitated proteins. Wash in batch for at least 2 hours with a

solution 1M NaOH,follow with a wash with 10 column volumes of distilled

water.

C. Elimination of strong hydrophobic interactions: Resuspend the resin in batch

with 30% isopropanol and wash for approximately 20minutes,follow with a

wash with 10 column volumes of distilled water.

D. Elimination of lipids: Wash in batch for 2 hours with a solution 0.5% of non‐

ionic detergent in 0.1 M acetic acid. Rinse away the detergent with

approximately 10 column volumes of 70% ethanol,follow with a wash with 10

column volumes of distilled water.

3. Add 5 volumes of 0.1M nickelsulfate hexahydrate.

4. Wash with 5 column volumes of distilled water.

5. Add 5 column volumes of the binding buffer. The column is now ready for use.

NOTE: If storing the column for a while store at 4°C in 20% ethanol.

If you don not need your protein to be in native conditions, try denaturing conditions such as 2-8M UREA. This is ensure that your HIS tab is exposed.

Do you know if your protein is binding to the column? You can test this by taking some of the beads/protein complex (just before elution) and run it out on a SDS gel to see if your protein is there. If it is, maybe you are having problems with the elution step.

How do you elute your protein? Are you using a column, or are you working in tubes. In any case, it may be good to incubate with elution buffer for a bit on a rocker before collecting the protein elution.

I do feel that you try TBS again. It might be making a difference, Else try what Kevin has suggested. Hope your buffer contains no chelating agents (like EDTA as a protease inhibitor)

If the protein was binding before, it could be that the load of Ni is low. HiTrap Chelating columns need to be recharged once in a while. The protocol is quite easy. First, you use a 0.1 mM EDTA solution to clean it up, then you wash the column with Phosphate buffer to remove any trace of EDTA. Recharge the resin with a 0.1 M Nickel sulphate solution, and finally wash it again to removed unbound Ni.

I hope it helps

PS: If you use protease inhibitor tablets, they should be EDTA free. Otherwise, the EDTA will remove the Ni and your protein won't bind.

try lowering the imidazole concentration to 200mM...works well for us

PBS does contain NaCl, MgCl & KCl and the net ionic concentration in the PBS could interfere with your protein binding to the column. If you are using atleast 2M Urea or GuHCl then use Tris or preferably Hepes buffer and elute with Imidazole. Avoid using Ca and Mg salts in the buffer.

Amrutha, recombinant proteins turns out to be trouble makers even in my lab at times while purification. I recommend you to start with a new column, reagents and crude protein preparation with a special attention into the buffers used in column and sample and its pH. You may prelong the incubation time of the column with sample in a rocker and use gradient concentration of imidazole starting from 50mM to 500mM. Sometimes Co2+ column works better for some specific proteins.

hi , Try regenerating the NI matrix .... your procedure is right and also run SDS PAGE to confirm Whether your protien is expressed properly or not ....

Try your purification using more amount of resin, may be 2 or 3 times more volume.

Try in presence of 5 mM imidazole, it should work if the column is properly regenerated. Washing in presence of 20-30 mM should help in removing contaminants. Your elution conc. is perfectly fine. Good luck

What expression system are you using? Are you using and ammonium sulfate cut prior to the resin? If you are, be sure you are adequately dialyzing out the salt before your resin column. Also, have you tried cobalt resin? I have seen some proteins bind better to cobalt. Good luck.

this is a very common problem with some of the His-Tag protein. first reason can be the His tag is not available properly due to folding of your protein. If this is the case try using mild Urea to denature it slighlty like 2 or 4 M. Second try to play with buffer like sodium phosphate, Tris, or hepes. Next is salt concentration use 100-500mM NaCl, high salt increases the specificity but sometimes hinders in binding. One try you can give with talon also as it has more specific binding than nickel. Good lick with you protein . hope this helps.........otherwise used denaturing conditions and then renature it.

What buffer do you use to elute the column? Using gradient could be helpful.

I guess your protein does not bind at all to the resin and it is also in the FT and washing. First of all, be sure that you are using a pH of 7 or 8, not lower to pH 7. Do not put any imidazol in the binding/washing buffer. I don´t know anything about your chromatographic procedure, but I would increase the retention time of the protein with the resine, for example binding in a batch mode for 1 h at very low shaking (at room temperature if it is possible) or reduce the flow rate if you use a chromatographer. If your protein doesn´t bind even with these conditions, use denaturing buffer with 6-8 M urea, batch mode binding, and perform the purification procedure by gravity or centrifugating the resin (with not more than 8000 g) and collect the supernatant.

If it is not possible at all, try first with another chromatography (i would suggest IEX depending on the protein pI of the protein) and afterwards try again with the Ni resin.

I have a series of questions, if you answer them systematically you might find the clue. Protein purification is a combination of science, art and luck--I think!. Now, what cells are these? Bacterial, insect, or mammalian? How do you lyse the cells and how do you exttract the protein? Is your protein suspected to bind DNA/chromatin (if so, use a nuclease during cell lysis/extraction and avoid EDTA/EGTA)? How long do you bind your protein to the beads? Where? How?What ratio of protein extract to beads? Do you have DTT/2-ME in your buffer Yyou should be wary of these)? How do you see your protein? By WB? If so, did you simply compare volumetric equivalents of input, bound resin and unbound supt? Did you see no binding to beads? If so, why are you doing elution? Do you have a linker between the last amioacid residue of your protein and the tag? Usually people use Gly-Ala-Gly (or a few more) as linkers. If you have none, the tag may be occluded by the protein. If you post a detailed description of your problem/protocol, it will be easier to address.

check that you dont have a signal peptide that might be cleaved leaving your protein without the His-TAG

If the protein is denatured, with urea,6M, and still does not bind, then you have a problem with many of the things described by everyone. This is easy to try. Sometimes the proteins just don't bind in their native state. So you can put the protein in urea with a reding agent( even 5mM DTT is ok), and then wash, and then slowly reduce the urea concentration, to about 1M, and then wash with 1 column volume of no urea buffer, and then elute. It shou ld work as your protein was soluble to begin with. But you can check first just to see if it binds in 6 M urea by adding it to some resin. If it does, then you can proceed with the washing and refolding on the column. Believe it or not, proteins with his tags, in their native state, don't always bind to the resin. This would be easier and faster to try than making a new construct that may or may not work.

As Anil said also, if you have EDTA or EGTA in your buffer, this could be your problem, so don't use it.