Can i simply wash with MilliQ to wash off the imidazole from the column in order to run the second set of purification of the same protein??
Dear Deepti
Generally i'm regenerating the colounm by stripping out the metal by EDTA and reload it.
Practically you can:
- wash the coloumn with 5CV (coloumn volume) of milliQ water
- add 5CV of EDTA 0,1M pH=8
Dear Deepti
Generally i'm regenerating the colounm by stripping out the metal by EDTA and reload it.
Practically you can:
- wash the coloumn with 5CV (coloumn volume) of milliQ water
- add 5CV of EDTA 0,5M pH=8
- wash the coloum with 10CV of milliQ water to remove the EDTA excess
- Add 3CV of NiCl2 0,1M pH=8
- wash the coloum with 5CV of milliQ! water to remove the EDTA excess
- equilibrate the coloumn with binding buffer
in this way generally i'm able to re-use the coloumn for the same protein many times with-out lost performances.
If you are using pre-packed coloumn is it important to load samples well centrifuged or filtered at 0,22uM because otherwise you risk that the coloumn will be clogged after several purification.
good luck
Manuele
Thanks Dr. Manuele Martinelli ! I highly appreciate for your detailed information regarding recharging Ni-NTA column. However, i wanted to know regarding the simple washing procedure that could be applied to reuse the Ni-NTA column for the second run purification of the same protein.
Dear Depti
generally this is the procedure that i follow also when i would like to perform a second purification for the same protein.
It is simple and quite fast, not stressful for the resin and and ensure to you that all the coloumn binding site are saturated by Nickel and therefore the maximun coloumn binding capability is retained.
Because all washing steps may remove some metal and you need to re-charge it for guarantee good resin performances.
good luck.
Manuele
Hi Deeti, I am not sure why you are searching for an alternative for the protocol which is tested ...resins wash away chemical after MES you just wash with water and then your buffer and you are good to go .... The alternative is wash out all nickel with 50 to 100 mM EDTA , wash with water and then add nickel to repack the column
Our general protocol for thorough Ni-NTA regeneration:
MilliQ H2O - 100mM EDTA - MilliQ H2O - 6M GuHCl - MilliQ H2O - 0.5M NaOH - MilliQ H2O - 2%SDS - MilliQ H2O - 100mM NiCl2 - MilliQ H2O.
Make sure the components 6M GuHCl & 2%SDS, 0.5M NaOH & 100mM NiCl2 do NOT meet. Or they make precipitate and clog the column and pumps and tubings.
Dear Peter,
Please add 20 mM Tris-HCl, pH=8.0, wash to buffer the system. Pure water would strip nickel ions in the long run.
Google Qiaexpressionist and download the pdf booklet. You will get everything there. It's the protocol manual from Qiagen that guides u in everything related with affinity purification using ni-nta.
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