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Hi Amala,

1. Did you verify the sequence or the presence of the His-tag? If you were not the person for cloning. I used to spend 14 months working on an clone and eventually I was allowed to check the sequence and verify the expression of His-tag by Western-Blot. There was no his-tag.

2. You can try ionic-exchange columns instead of Ni-Column. In this case, chaining the cationic column and anionic column in a linear way. You target should either stay in 1) cation or 2) anionic or 3) flow through. You can verify the presence by SDS page.

3. What's the temperature you used for purification? I.e. if the pH 7.5 of tris was under RT, then it should be 8.3 in cold room.

4. Increasing the concentration of Na+ will increase protein solubility and decrease non-specific binding ( possible langmuir adsoprtion). Normally, people use 200-300 mM NaCl. If the Tris-tri sodium is used, the concentration may need to controlled < 50mM. For instance, if 50mM Tris-tri Na was used, then 150mM NaCl could be introduced in the buffer system additionally.

5. 10mM Imidazole is used for removing non-specific adsorption in this case.

6. In some cases, everything looks normal, but the protein just does like to bind to the Ni(Co)- column. In this case, using ionic-columns will be an alternative method before you try another vector or expression system.

The advantage of ionic-columns is you can buy the beads (+ and -) from sigma with much less cost. You even don't need to use an AKTA system.

I hope this will be helpful and see your publication soon.

Kevin

On Mon, Aug 13, 2018 at 6:50 AM amala mathimaran <[log in to unmask]> wrote:

Dear All

I am working with HIS – tag protein in N-terminal (hexa histidine). The protein from Prokaryotic origin cloned into pET30a+ vector and expressed in E.coli BL21 cells. The expression was good. I am trying to purify a protein using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same as buffer A but 250mM imidazole). I eluted the protein in step wise gradiant. the protein was less binded in Ni affinity IMAC column because eluted fraction contain less amount and the protein remain present in the Flow through. Can any one suggest how to increase the binding affinity of the protein and how to purify the protein. The protein PI was 6.33

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