I have pRSET_A vector fused with human Prdx6. My vector has enterokinase cleavage site inorder to separate his-tag. By using enterokinase ,i have plan to remove His-tag from my protein.Now i can't understand how to separate enterokinase and my untagged protein? My protein is 25kDa protein and enterokinase is 150kda protein.
Hi there, So your eluent have both; the protein of interest (25 kDa) and the enterokinase (150 kDa). To separate them you will be needing to do a size exclusion chromatography (aka gel filtration chromatography) which separates biomolecules on the basis of size. It would be better to use 200 GF column (200 kDa cutoff), through which u must get two peaks,one of your protein and other for enterokinase, at different elution volume.
Hope it helps!
Hi there,
An easy way to remove EK is to use His tagged EK: just by passing the digest onto NiNTA you remove both EK and non digested protein. Otherwise the use of EK removal kit might also be considered:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/prkebul.pdf
Hello, I have a protein fused to Trx-his tag, cloned in PET32, I would like to remove the tags (trx and histidines) , my protein is 14kDa and the fused aprox 30kda, how can I remove the his tag by EK and to also eliminate the enterokinase from my prep? It is possible to digest on resin and to eliminate at the same time the enterokinase?
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