Hi Sudheer, seems your column became oxidized, possibly 0.5M NaOH was a little too harsh? (broke the complex and precipitated the Ni as "hydroxide" which then got oxidized by oxygen dissolved in the solutions?)
Anyway, flush your column with a few ml of 0.1...0.5M EDTA (removes Nickel), water (removes EDTA) and then re-charge it with a few ml of 100mM Nickel chloride or sulfate, then again water and then your buffer of choice. Please remember Nickel salts have some carcinogenic and allergenic potential and that Ni is a heavy metal that likes to be disposed of properly. If the colored stuff does not disappear with EDTA initially, try water, 100mM HCl, followed by water, first. HTH Wo On Thursday, November 15, 2012 11:27:19 PM UTC+8, sudheer sangeetham wrote: > Hi everyone > > > > After elute the protein from Ni-NTA beads I washed the beads with 0.5M NaOH > > to get rid off imadozole and traces of protein on the bead, but > > unfortunately the column turns yellow colour? My recombinant protein which > > fusioned to m-cherry, even after elution the column looks light pink in > > colour. could anyone explain why it has happened and how I could get back > > Ni-NTA beads to normal light blue color. > > > > Thank you in advance > > > > -- > > Sudheer Babu.S > > Research Fellow > > Institute of Biochemistry > > Biological Research Center > > Szeged,Hungary. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
