You can run the denaturing solution over the column until the column is clean, then wash it out using the usual storage buffer. Make sure to use plenty of washing buffer to remove all the denaturant.

Dear Tatcheewa

an other possible reagent to regenerate nickel beads is edta. you can strip nickel from the coloum with edta..washing with water and add again nickel in solution.

good luck

Manuele

Urea needs time to denature and dissolve aggregated protein. Leave the column overnight with the urea solution not running and use 8 M solution. Removing nickel ions will only work if the ptotein is strongly attached but not aggregated. You can also use naoh (for this remove nickel ions first) or a surfactant solution for faster cleaning.

Adam B Shapiro Manuele Martinelli Omar Gonzalez-Ortega

Thank you for your useful suggestions.

Hello

If you need to clean your column ,you can add urea (8 M) & close your column from running ,this will denatured all proteins attached to the column ,then you wash your column with the buffer as you need.

Brown Ni resin can also result from use of reducing agents.

Easier and quicker is to strip of the nickel with EDTA and clean the column with 1M NaOH. Collect the nickel and dispose appropriately.

Dear Thatcheewa Apichatayanon, dear Maria,

brown or orange nickel is probably due to the use of reductants like DTT or ß-mercaptoethanol. You can easily regenerate nickel by using acetic acid. There is a nice overview of regeneration protocols (wash after every run, wash and regenerate with EDTA/NaOH, wash and regenerate after use of reductants) at

https://cube-biotech.com/media/pdf/08/34/42/Washing_Regenerating_HisAffinityResins.pdf