Whenever I purify the His tag protein plasmid DNA is bound with it & troubles in assays, though purification with His trap & anion exchange column was done, yet DNA is bound to protein. How to get my pure protein without any activity loss for my assay with the protein. PI of protein is 4.5
Have you tried to add DNAse to cell lysate? or at least perform DNAse treatment at some point?
If DNase treatment doesn't work, you could try including a high salt concentration (e.g. 0.5 to 1 M NaCl) during the IMAC or DNase treatment step to partially denature the protein.
Even after 500mM Nacl wash DNA is bound to protein ! Thanks for your suggestion
you can go for polyethyleneimine precipitation or I would suggest to do the following: you add some DNase already into the cell lysis buffer (this also makes your cell lysate less viscous). As an optional step after cell lysis you can add LiCl at a final concentration of 1M to precipitate the DNA. A centrifugation step (more than 25,000g) will clear the lysate from any insoluble molecules, such as the precipitated DNA, cell wall fragements and protein aggregates. In the optimal case your protein carries an affinity tag which allows you to perform a first affinity capture step. Then you purify the protein with the heparin column. Afterwards you can do a polishing step employing size excusion chromatography
It's interesting that your protein is bound so tightly to DNA. Is this consistent with the function of the protein? Is it possible that there is a covalent bond between the protein and the DNA?
yup! protein bound to DNA..! It has function with DNA.. But without removing DNA assay are not conclusive & We employ 1M Nacl to precipitate DNA
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