I am doing work on in vitro expression of protein. My protein has been expressed along with his-tag which i am unable to purify with Ni-NTA bound column. Whenever i load my sample the protein elute out in wash buffer. This may be happening as his-tag is inaccessible or the column beads are not working. Kindly suggest the solution. I may treat the protein with urea for which , i don't know the protocol. Kindly suggest.
Uploading my SDS-PAGE gel photograph
right to left
Lane1- Marker
LAne 2- supernatant of protein expressed
Lane 3- Pellet collected by centrifugation redissolved in buffer
Lane4- flow through collected after sample loading
Lane 5- wash buffer
Lane 6,7,8- elution buffer
Alrights, lets have a look at this:
Yes, it may be that your His-tag is not accessible by the beads. TO overcome this you sadly have to edit your contruct slightly. Either add the His-tag to the over terminus or add a small spacer sequence 6-8 amino acids between the end of your protein and the current position of the Tag. Bascically "expose" the tag for the beads.
Another reason could be your buffers. Are you using premade buffers of a Kit or prepare them yourself? Chelators like EDTA/EGTA or reducing agents like DTT can more or less render your Ni-NTA column useless if you have them in one of your buffers by mistake.
So either check your buffers or use an alternative to Ni-NTA that tollerates higher concentrations of such substances. Our Ni-INDIGO columns are an option here:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/
Have you confirmed whether the induced protein along with the His tag is being expressed by Western Blotting with anti-His ? There is a chance your insert may not be in frame with the plasmid's ORF. I am suggesting this because it is very unlikeley that your protein did not bind to the bead at all. Ni-NTA beads are very sticky and also have a tendency to bind to a lot of non specific proteins, even in lysates with abundant His tagged proteins. If you think the construct is fine, then you may need to revisit bead preparation before binding. Make sure all the alcohol is washed away and the beads are properly equilibrated in the binding buffer before use.
Your protein is not bound at all, it may be that the His-tag is not accessible by the beads. You may need to edit your construction, or try batch purification with a longer incubation time of your protein and your resin. This may work for you.
What is your buffer composition? It looks however than a lot of contaminants are able to bind to your resin, so it's difficult to say that your beads are not working.
And are you loading the pellet or the supernatant on the column?
As everybody has given nice explanation,acc to me you just try by increasing the amount of ni nta beads as well as do not add imidazole in lysis buffer,may be this could help you
Hi,
Is there a TEV cutting site between your target protein and His tag? Or whether to use antibodies against His for WB? I would say that you should first prove that the tag is indeed expressed, and then check the column, buffer, and operating procedures.
Roger Davis I face the same problem when purifying the protein. My target protein is 6His-MBP-TEV-XXX. I am wondering whether the TEV cutting site or other factors influence the protein binds with beads?
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