Dear friends,
Does anyone know how can I re-charge HisTrap HP 5 ml column?
I use HisTrap HP 5 ml column to purify proteins and since I used it multiple times I had to re-charge it but it was not successful.
Hers is the steps that we followed:
Do you inject NiSO4it by syringe, AKTA, or any other devices?
Looking forward to hearing from you soon.
Regards,
Sara
I do not understand the overflow part. Anyway, you have to wash the system with water to remove edta before injecting nickel ions.
The device is irrelevant. You can run injections or pump directly.
For the standard GE nickel columns, your EDTA wash sounds good. Wash the column several times with miliq H2O to remove any EDTA. You can also try using 100 mM NaOH or 4 M GdHCl to get rid of any junk proteins that are still stuck in the column. Then repeat water wash for several column volumes. Then recharge column with Ni2+ SO42-. You can do all this with a syringe and syringe adapter.
Dear Sara Heidarpanah
peristaltic pumps could be used instead syringes or akta to equilibrate, was regenerate and perform purification with HisTrap HP 5 ml coloumn as you can briefly see in the attached video.
The fact that the Nickel solution make overflow differently to the other is quite stange because if the nickel is well dissolved there is no reason why the pressure of this solution is different from the others.
Is it your solution clear and did you filter it at 0,22um? I suspect that the problem is not your loading approach but your nickel solution.
Best regards
Manuele
I agree with those who commented above: wash with water before loading with your Ni-SO4 solution, filter all solutions before applying to your column.
Just let me add that you should avoid to introduce air bubbles when you use the syringe as it may provoke some trouble.
I usually do the stripping and re-charge with a syringe and it works perfectly fine. Make sure to use syringes that you can screw into the adapter provided with the column.
Since you managed to strip the resin I would exclude that there is clogging caused by some precipitation (unless you didn't wash off the EDTA after stripping).
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