If you are using C terminal his tag, then try N terminal one(ie, change the construct).

As Gaurav stated, high concentration of NaCl can eliminate the interference of DNA, but it can not guarantee your protein will bind the resin.

You can try to change the binding condition, detergent, but I strongly suggest you try other construct simultaneously.

Hi Natalia

Some times the His Tag on your recombinant protein may get 'internalized' during folding...as a result it owuld not possible for such a protin to bind to Ni-NTA column during purification.....we had also recently seen such an example in our lab....You can denature this protein and then try to bind to the Ni-NTA column and then do 'on column folding' to get the folded protein....it works many times

bye

Hi, we have already tried with denaturing buffer (using guanidinium), and still can´t get the protein.

Hi

May be you can check with some other denaturant like Urea...and also more important .....is your cloning perfect.....do your protein have His tag....if you have not verified it....isolate the protein from inclusion bodies (if it is getting there)..denature the protein and do a western with 'anti His antibody'.....go sequentially.......if it is still not possible....try to change the location of the His tag....on your protein

bye

Hi Natalia,

I agree with Ajay's comments, ans steps to deal with your question are :

- Are jou sure of your plasmid's sequence (Frameshift mutation...)

- Did you perform protein production tests to know if your protein is expressed or not, and if it is soluble or insoluble ?

If your protein is insoluble, this can bury the Tag and cause impossibility to bind on NiNTA. If the Nter of your protein is "naturally' buried, this also can bury the Tag... But anyway, if it is expressed, there is no reason why it shouldn't bind to NiNTA on denaturing conditions (7M urea normally does the job wery well...).

We checked the His tag protein expression, using anti His-antibody in the soluble fraction. That works OK.

- We can try the purification with 7M urea

In other web pages I read that increasing ionic strength (1M NaCl) in the lysis buffer may increase the yield of the purified protein. Has anyone tried this protocol?

Thanks for the advices!!

Natalia

Hi Natalia,

how do you break your cells? Sometimes people use lysis buffer containing EDTA ;)

You could also check with another His-tagged protein, if your resin is OK.

Good luck!

We have purified other His-tag protein without any problems. The resin is working fine.

We don´t use EDTA in the lysis buffer, but we can try.

Thanks

Natalia

Don't use EDTA with Ni-NTA, this would elute the nickel from the column !

There are different variation you can try to make the protein binds th NI-NTA column. As in my case i tried high concentration of Nacl in Wash and elution buffer. You can also try 1XPBS (it gives more soluble fraction) and Phosphate buffer(pH 8)..i tried my protein with 1XPBS and it works..just you need to optimize it nicely..

All the very Best Natalia...

If you are using C terminal his tag, then try N terminal one(ie, change the construct).

As Gaurav stated, high concentration of NaCl can eliminate the interference of DNA, but it can not guarantee your protein will bind the resin.

You can try to change the binding condition, detergent, but I strongly suggest you try other construct simultaneously.

I agree with the previous answers. Did you check the DNA sequence of your clone? probably the number of histidines it's not enough.

Had the same problem; if it is not binding it might be buried in the structure of protein since the western blot is working. I would try to go for IEX chromatography if you know the pI of protein (or you can get the theoretical one using Expasy protoparam) and see if you are able to purify it. Or you could also use urea (low concentration) to slacken a bit the structure to expose the his tag or also Cobalt instead of Nichel (it has much higher affinity than nichel). Hope I helped.

the denaturing conditions option would be in my opinion the last thing to try. To refold a protein is not so easy! ;)