Do realize that affinity purification can only go so far; If you are loading a relatively crude preparation where your protein is only a minor species (a likely scenario if you are using a plant host) you cannot expect a pure protein to come out at the other side. You might try something cheaper and of higher capacity first (i.e. ion exchange) and then use Ni-NTA to polish the result.

Hi Reyazul,

if your protein can take low pH, you can try if you contaminating bands still appear after elution with low pH (5,3 to 4,5). This will lead to protonation of the histidine residues. To protect your protein you could collect it in tubes containing a 10x concentrated buffer with neutral pH, to keep exposure to low pH as short as possible.

If the unspecific bands still apear and if they migrate at a lower molecular weight region in your SDS-PAGE you might want to test if these are degradation product of your protein of interest.

If you are not using a chromatography system which would allow a gradient elution you could try using a step-elution protocol with 50, 100, 150, 200 mM Imidazole e.g. and monitor all fractions for contaminating bands and protein of interest.

There are many other options, but I will leave it like this and suggest that you might also check:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909483/

http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAexpressionist_EN.pdf

Best,

Kai

A co-purification of E. coli proteins is quite usual, very often in His-Taq purification (my own experience, see also: Protein production and purification: Nature Methods; 2008, Vol 5 No.2 p135). You can try taq cleavage after the first purification and than do affinity purification again, your protein will not bind anymore, but the undesired should. If the other protein is a chaperone, it will be more difficult.

Another option is to use a different expression strain, for example NiCo(DE3) from NEB was especial developed for this kind of question. (https://www.neb.com/products/c2529-nico21de3-competent-e-coli)

You can either try stepwise elution and compare the fractions for their content of your protein and contaminants or re-purify on another column, either gel filtration or some ionex should work.

Do realize that affinity purification can only go so far; If you are loading a relatively crude preparation where your protein is only a minor species (a likely scenario if you are using a plant host) you cannot expect a pure protein to come out at the other side. You might try something cheaper and of higher capacity first (i.e. ion exchange) and then use Ni-NTA to polish the result.

@ Alejandro

Actually, you want to use affinity first an then polish with another column later since the yield is low and capturing seems to be an issue. But yes, there is only "that much" a column can do. The price of the matrix doesn't really matter since Ni-NTA and the most common ion exchanger resins can be used at least 100 times, if handled and stored properly.

@ Md

Make more cells and develop a two or three (four? eight?) step protocol to purify to homogeneity. If yield is your concern then add more crude in the beginning. Getting more pure protein always means losing more protein. If you want protein so pure you can't trace a single contamination on a silver stained gel, get friends with the idea of throwing >90% of it out during purification.

Also, try not to dilute your lysate too much since it will reduce binding of the his tag to the matrix. Alternatively, concentrate it before adding it to the column.

Also, what Kai said was really good.

Thank you all for your valuable information regarding protein purification. I got lots of idea through out your comment and i should try those. Thank you again.

@ Stefan,

Sorry, I don't get your idea. Would you explain a bit more?

You do have a point regarding the price and reuse of Ni-NTA, but it is valid only in academic/research labs. It won't take you very far while arguing to a GMP auditor in industry.

The order of columns is questionable. I was taught as well that affinity chromatography should be last, but when I was reading some GE Healthcare manual recently, they recommended to use AC as first step.

@ Alejandro

The first step serves primarily to capture as much of the protein as possible which is best done by affinity purification. If the concentration is low in the lysate you should always use affinity chromatography first since ion exchange captures not only the target but also more contamination (e.g. DNA if you haven't got rid of it). That all goes on cost of the superior capacity which is also not that much higher (or even lower) than Ni-NTA, depending on what you use.

You always want to eliminate as much dirt as possible in each step. This is how I learned the purification procedure (from a guy working in industry for 30 years BTW):

1. PEI precipitation (get the DA out)

2. AmSO4 precipitation (cut your protein out)

3. Affinity or Pseudo Affinity chromatography (get the most target out)

4. Ion exchanger I (weak or strong depends on your problem)

5. hydroxylapatite (optional, concentrate sample)

6. ion exchanger II (the other one, see 4)

7. SEC (fine polish the product)

@ Tomáš

affinity last step? I can not think of a valid argument for running a sample over affinity column post SEC. If your affinity column can still add to the purity at that stage, you used the SEC prematurely and got suboptimal performance from it.

@Stefan

Then we agree that you should use high capacity, low selectivity methods first. My problem is how do you reconcile that with putting affinity chromatography before IEX. How much target AC will 'get out' will depend on the number of competing, low-affinity 'nonspecific' interactions (high in the case of a plant homogenate) and the affinity with the target (not that high in Ni-NTA). Why not clean the sample first then with something cheaper and of higher capacity than AC? We may argue over whether IEX fits that bill or not, but I've had my share of cases where unsuccessful attempts to purify low-abundance species from crude homogenates by Ni-NTA have been solved by fractionating the sample first by IEX.

And put it as you may, but most IEX resins have higher capacities and are cheaper than most Ni-NTA gels.

For the price, you buy those matrices anyway and keep good care of them, so the order of usage doesn't really matter in terms of dollars.

For the order of usage, you get less contamination from Ni-NTA than from IEX because the target interactions are much more specific. I never encountered weak binding of His tags to Ni-NTA but you should be able to overcome that problem by adding a 12xHis tag instead of a 6xHis tag and use more imidazole in binding.