Hello everyone,
I am doing protein purification using His60-NiNTA column,
Once i used and at last i regenerate that column as per said in manual with 20ml equilibrium buffer following mq water and 20% ethanol and stored it at 4 degree.
two time i used this column, but maximum how many time we can use this same column for same protein purification.
Dear Tulsani
if you are corretly stored it (4°C in etanol 20%) and regenerating well the coloumn, by stripping metal with EDTA and reload it, you can re-use it many time.
If the nickel when is added yo the coloumn is able to confer an homogeneus green colour which means that all the resin is still functional and able to bind the nickel, and the conterpressure of your coloumn do not increase a lot the coloumn is ok.
Generally i use a coloumn for a single protein but also for long times and i change the coloumn when the conterpressure increase and i'm not able to reduce it by coloumn regeneration by stripping metal with EDTA or remove protein precipitates with a buffer containing UREA 6M.
good luck
Manuele
Dear Tulsani
if you are corretly stored it (4°C in etanol 20%) and regenerating well the coloumn, by stripping metal with EDTA and reload it, you can re-use it many time.
If the nickel when is added yo the coloumn is able to confer an homogeneus green colour which means that all the resin is still functional and able to bind the nickel, and the conterpressure of your coloumn do not increase a lot the coloumn is ok.
Generally i use a coloumn for a single protein but also for long times and i change the coloumn when the conterpressure increase and i'm not able to reduce it by coloumn regeneration by stripping metal with EDTA or remove protein precipitates with a buffer containing UREA 6M.
good luck
Manuele
The His60 Ni Column can be reused 4 times to purify different preparations of the same protein.
Hi Tulsani Nilam . In theory you can use the same column resin indefinately. These columns have metal chelating -COOH and -NH2 groups that can take a lot of punishment.
You can clean them like any other ion exchange resin, e.g. DEAE.
https://www.sigmaaldrich.com/technical-documents/protocols/biology/ion-exchange-chromatography/appendix-10.html
Even if the backpressure increases and cannot be brought back by washing with urea, you can simply empty the gel into a beaker, wash (do not use a flea for stirring, as this would break the beads), decant the fines, and then repack the column.
When properly handled, these gels are near indestructible.
HI Tulsani, as long as you keep your column in good condition you can use it for a long time. Be sure that your sample does not contain particles as backpressure can build it. I would always strip the column with EDTA buffer to remove all protein, afterwards you can reload with Ni
GE Healthcare sells pré-cast columns (1ml or 5 ml) , these columns are very simple to use and gives very good results
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