I alwasy use Ni-NTA to purify 6xHis-tag protein,my buffer usually is netural PH with low beta-ME (1mM-4mM)or NO reducing agent , after one time use the column fade and alway become little yellow and cannot binded my protein ,I have wash colum with 6M guanidine hydrochloride，then1.5M Nacl next 0.1M NaOH then 30% isopropanol finally wash with Water.but the column still cannot become blue as new one and cannot bind protein efficient as first time use. I still try to use EDTA to chelate Ni and recharge with 0.1M NiSO4, but the column even cannot tolerate 1mM beta-me and with low protein bind capacity. Did any one have some good suggestion for how can improve the column binding capacity.(the bacteria lysis after 18000rpm centrifuge for 1hour then filtration with 0.2uM filter membrane before load to column )
Good day, hopefully I am not to late to give you help.
Ni-NTA can indeed be regenerated & reused multiple times. Sadly I cannot give you solid information on how to do it with His Trap Ni-NTA. I do not know how its core is made up and which reagents ist tollerant to.
You can try the washing & regeneration protocol for our PureCube Ni-NTA agarose: https://cube-biotech.com/washing-and-regenerating-his-affinity-magbeads-and-agarose-resin
Maybe it works his His Trap Ni-NTA, I just feel not confident enough to "garantee" it.
However it works garanted with this Ni-NTA agarose: https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/ni-nta/179/purecube-ni-nta-agarose
We offer Free samples if you are unsure: https://cube-biotech.com/us/campaign/index/emotionId/45 .
Hi,
The main reason behind the color change should be the oxidation reaction of Ni ligands of the column.
I can suggest only the common advices below, I sadly see that you had tried them, but higher volumes of washing and higher concentrations of stripping and equilibration buffer may help;
- · Do not use high pH buffers,
- · Do not use Reducing agents (BME, TCEP or DTT) even at low concentrations.
- · Strip column with EDTA and re-equilibrate with NiS04,
- · Use always fresh buffers.
- · Be aware with higher imidazole concentrations pH will shift to a higher value, readjust to neutral after imidazole addition and always prepare fresh,
About binding capacity I need to ask additional several questions;
- What is your loading buffer composition,
- Are your his-tags are stable and can you confirm them with W.Blotting?
- Which concentration protein sample are you loading to the column and are you also determining your target protein at flow-through fractions?
- After how many injections are you observing this the non-binding issue?
Best regards,
Emir...
Your columns are indeed a little bit weird, is the sample you are loading very visquous? How many mL of sample do you load each time? Anything else in the buffer?
Do you use the column often? After how many uses did you observe this issue? How do you preserve your column between each purification? Any issues with air bubbles or a long time without stoppers (inlet and outlet)?
Hi Liao,
First I would like to tell you that HisTrap FF columns that you have on the photos are not packed with Ni-NTA resin. They are packed with Ni charged Sepharose Fast Flow and don't use NTA for attaching the Ni ions. This resin is not compatible with any EDTA and/or DTT in the sample and/or buffers as these agents with drag the Ni ions out from the resin that will become white (as you have seen) and you will lose binding capacity (as you have seen). There is a constant product development from several suppliers to come up with resins that are resistant to addition of DTT and EDTA when doing purification of His-tagged proteins that need these additives to be kept in an active form. I want to tell you that the company I work for now has just launched a new resin WorkBeads NiMAC (as available in prepacked formats as BabyBio NiMAC 1 ml and 5 ml columns) that you can use with up to 20 mM DTT and 20 mM EDTA in the sample/buffers without losing any binding capacity and the resin can be used many times. If you are interested you can contact me, [email protected]
Best regards, Anna
Thank you all, your advice is very helpful, I resolve the problem with replacing a new Ni prepacked column, GE healthcare HiTrap Excel 17-3712-06 .and this column is better than above I mentioned, but also much expensive.
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