Good morning,

According to your text, you are expressing a protein with a His Tag.

1) Are you sure that your His tag is expressed. Did you do a western blot with a His tag antibody?

2) Where is your His Tag (N or C terminal position)? The fact that your protein is eluted with the washing buffer may explain that the binding between the tag and the column is weak. The conformation of the protein may avoid the correct binding of the expressed protein. If you change the position of the tag you could have a better retention.

Good luck

Johan Gardères

Good morning,

According to your text, you are expressing a protein with a His Tag.

1) Are you sure that your His tag is expressed. Did you do a western blot with a His tag antibody?

2) Where is your His Tag (N or C terminal position)? The fact that your protein is eluted with the washing buffer may explain that the binding between the tag and the column is weak. The conformation of the protein may avoid the correct binding of the expressed protein. If you change the position of the tag you could have a better retention.

Good luck

Johan Gardères

As Johan Gardères said, have a go with the His-tag on the opposite end of the protein if you are sure of the presence of the His-tag in your first construct.

As well....you may want to check the pH of your protein to make sure it is at the right pH for the Ni-NTA column (pH 8 I believe).  The pH would also affect the binding interaction between the Ni an the His residues. 

hello dear John.

you're right, my protein has his tag.

before I could purify my protein in high concentration, but recently my protein hasn't been purified. is it possible their conformation change suddenly?

my Ni-agarose resine is new and I charge it one time.

I use Glycerol 10% in elution buffer for protecting my enzyme from high concentration of imidazole ( my enzyme lost activity after 2 saat in elution). after using glycerol, when I wanted to purify my protein, they eluted in wash buffer. is it possible glycerol destroyed my resine?

Not to beat a dead horse but this can be fixed by ensuring that you are purifying protein from the correct clone (with His tag) which bound to the Ni-NTA before. It is hard to believe that there was a conformational change that occludes the His tag but a simple way to check this would be to put a small portion of the protein in 8M urea and see if it binds the resin then.

I concern that what kind of resin you used. The protein is easy to wash out by low concentration of  imidazole. 

Do you actually see the protein in the wash buffer?  Or does it just not come out in the elution?  (are you sure it's binding to the resin in the first place?)

I've had nickel resin go off before.  You can try a cobalt-based (TALON) resin.  That tends to work better in my hands.    

This sounds silly, but your resin is still blue, right?  If it goes white, that means the Nickel has come off (Cobalt resin would be pink).  

Glycerol should be ok - My TALON resin manual says it should be ok with glycerol up to 20%. 

A few more details would be helpful:

Is the 6xHis tag on the N- or C- terminus of your protein?  What are the components of the loading, wash and elution buffers (include pH; pH 6.0 or lower might contribute to protein elution)?  Any DTT (>1 mM) or EDTA in your buffers ('Can't have those when using in IMAC columns)?  Is your protein truly eluting during the wash, or is it simply flowing through the column and not binding in the first place?

I believe your problem lies in the answers to one or more of these questions...

You say that you used to be able to purify your protein to high concentration. I presume you mean you had high yields but you haven't purified it for a long time. So are you expressing this again or purifying from old pellets? When you are expressing are you starting from a glycerol stock, streaking a plate from a glycerol stock and starting with a drag colony or retransforming. If you know this has worked well in the past I would go through the steps to see what has gone wrong. Do your cells still contain plasmid? If you run the lysate on a gel do you see overexpression of your protein? If not, make sure your cells contain plasmid. Best thing would likely be to start fresh. Do a retransformation of your cells with the recombinant plasmid and see if you can replicate your original expression and purification. Take aliquots at each step to ensure your protein is present and where it is present. You can do a quick western with an antibody against the His tag to ensure the tag is still there. If it is, try stripping, cleaning and recharging your resin.

Best of luck!

You told that the your had a positive clone from which you could purify your protein. Did you also use glycerol to purify it? (glycerol provokes a lot of hydrophilic interactions)

Overwhise make sure that you construct is still available by doing a PCR to check the size of the insert and the sequence? It can happen in bacteria some recombinaison or spontaneous mutation that can modify the sequence or change the ORF.

Good luck

Johan

You can try elute your protein in denaturing or hybrids conditions. By using Urea in buffers system. This is a good alternative to try it. After that, you can dialyse your protein against any buffer you choice.

You can try and change your buffer capacity so that the molarity of the eluting buffer will be higher than that of the wash buffer. The physiochemical parameter of your protein of interest must be put into consideration in your choice of buffer. 

As Johan said, if it is being expressed, your protein just isn't binding the column efficiently.  As he mentioned, changing the position of the tag may make it more accessible.  Also, your protein could be misfolding; you could try to increase the amount of soluble, folded protein by changing the expression conditions (lower amount of IPTG (if you're using a T7lac promoter), lower temperature, but for a longer time).  This may also help your tag to be more accessible and bind the column.

Another strategy you could use would be to do extensive washing without imidazole to get rid of some of the extra protein, elution with elution buffer, and then an additional purification step.  For instance based on your proteins characteristics, you could use ion exchange or size exclusion.  If you have a cleavable his tag, you can cut it off and then rebind to the nickel column - your protein should no longer bind the column, but hopefully the other proteins will (this is never perfect, but it might help).

I don't know  what is your protein or its MW. each protein has its unique purification method.  Try gel filtration or ion exchange chromatography or ammonium sulfate precipitation for first step. then you can use affinity. Study previous works on your protein.

try increasing the salt concentration of your buffers and make sure the pH of said buffers are correct. 

Unbind protein will also elute out with 10mM imidazole because it is also compete with his tag.