I have a problem with my protein purification, I'm using the Ni column chromatography, running my cTnI protein after cleavage from the His tag to get the protein without the His tag, but it's leaking, I can't get a pure protein. Can anybody help? Thanks
Hi!! Zahran, Can you write in detail about the procedure you following for your purification. I can suggest you in detail...
I produced cTnI connected to PagP (a membrane protein) and the His tag, after cleaving the protein from PagP-His6, I need to purify the protein by running it through the column, but for some reason, the His6 part leaks with the protein in the flow through and with the binding buffer. It's 100% cleaved as shown in the gel. Thanks for the help
Okay You try these things.
First you incubate your cleaved fraction with NI-NTA in column for 30 mins at 4 Degree.
Second you can use 20 mM phosphate and 300 mM Nacl buffer for your purification, here you can increase the concentration of Nacl till 2 M to increase the ionic strength in results binding of His tag to NI-NTA will increase.
Third if your buffer composition is different while cleavage reaction you can dialysed your protein in the above mentioned buffer then proceed for purification.
Fourth Slow down your flow rate while purification.
Fifth do not wash your column because here you are expecting your protein in wash fraction. So take less amount of buffer and wash your column there you get your target protein.
Try these things, still you don't get the results i'll try to suggest some more points later.
Good Luck.
The Ni-column is used to capture the HIS-tagged protein, it is the main purpose, if you remove the HIS tag then the Ni-column does not work.
Yes, that's why I want to get my protein after removing the His tag in the flow through, even though, the cleaved His tag leaks with the protein
What are your buffer conditions? What is the residence time on the column? What do you mean by leak - does a small fraction of the fusion partner leak or all of it? You need to be a little more specific to allow us to give useful suggestions.
so it's cardiac troponin I, cleaved with Ni from the fusion partner, I added EDTA to get rid of the Ni, then dialysed it to get rid of the EDTA, and dissolved the pellet in 6M GuHCl.
I got the fusion partner leaking in the flow through, so obviously, the His tag that's attached to the fusion partner isn't binding to the column.
Thanks for helping.
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