Hi,
I purified a protein (about 28KD) via the following steps:
1- Firstly, I used buffer A native(Qiagen)-NaH2PO4,NACL,Immidazol,ph8
2-then after centrifuging and separating my pellet , I added buffer B denature(Qiagen)NaH2PO4,Tris-base,Urea,ph8 and washing it for 2 timesNaH2PO4,Tris-base,Urea,ph6.3
3- Then I eluted it 5 times-NaH2PO4,Tris-base,Urea,ph4.5
The result shows that I have very sharp band in my flow through while the elut bands were very weak.
How can I remove the flow thorough band and have sharp bands in my elut?
The photo of my result has been attached
This may not be buffer compositions for qiagen Ni-NTA columns, but for ours (Cube Biotech).
But since its Ni-NTA nonetheless it could help.
https://cube-biotech.com/us/purification-of-his-tagged-proteins-under-native/denaturing-conditions-using-purecube-his-affinity-magbeads
If that does not work for some reason, I recommend your to try our Ni-NTA colums instead:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/ni-nta/184/purecube-compact-cartridge-ni-nta
Looking at the amount of protein you have in your flow-through it seems that your protein is not binding to the resin very well. When dealing with an insoluble protein I usually try several different denaturing agents, and renaturation strategies. There is a vast amount of literature on this subject. The pH on the other hand will influence binding on the Ni-NTA resin, so you also need to take this into account.
If you just need a little bit of protein for your experiments, you can collect what you have now and concentrate it on a centricon.
A very simple thing to try is to rebind your flow through to fresh NTA resin, I have found that this worked very well for one protein I was purifying under Denaturing conditions.
Is it possible that you are not using enough resin? What is the volume of your elutions? Are you sure that your protein is entirely insoluble?
you have to check the location of the histidine tag ,if its in C terminus try to clone it in the N terminus which is better for target protein purification in many cases ,also you have to check your Nickel column if is charged well or not as in some cases it’s need to fresh charged to bind to the protein of interest.
@ Vince Murphy
Hi
I use 400 micro Ni-NTA for pellet and 150 micro washing buffer and 150 micro elution buffer.
I purify with Centrifuge ,donnot work with Nickel column
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