Ni-NTA Resin(NN01-RA091)
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Description : Ni-NTA Resin is nickel ion (Ni2+) charged agarose, to which Nitilotriacetic acid (NTA) is coupled. This coupling form is a very stable octahedral structure of nickel ions in the center, which can protect the nickel ions from attack by competitive small molecules. This resin interacts with the histidine, cysteine, and tryptophan on protein or polypeptide, effectively separating them from a sample.
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Specifications :
Protein Purification Protocol :Buffer preparation
All water and buffers are recommended to be filtered with a 0.22 µm or 0.45 µm filter prior to use.
Equilibration Buffer：20 mM Na2HPO4, 0.5M NaCl， pH 7.4
Binding buffer：20 mM Na2HPO4, 0.5M NaCl、20-40 mM imidazole， pH 7.4
Note: Optimize imidazole concentration as needed for target protein binding
Elution buffer：20 mM Na2HPO4, 0.5 M NaCl、500 mM imidazole, pH 7.4 Imidazole concentration range: 250 mM-500 mM.
Sample preparation
Protein samples should be centrifuged and filtered before being loaded on the column to avoid the accumulation of solids on the column, which will affect the life of the column.
Purification Procedure：
1. Perform all steps at a flow rate of 0.5ml/min for a 1ml column, or 2.0ml/min for a 5ml column.
2. Wash the resin with 5-10 column volumes (CV） of distilled water.
3. Equilibrate the column with 5-10 CV Equilibration Buffer until the baseline is stable.
4. Load the sample at the flow rate outlined in Step 1.
5. After loading, wash the column with Equilibration Buffer until the baseline is stable (OD280 is at baseline).
6. Wash the column with 5-10 CV of Binding Buffer. Collect the unbound sample.
7. Elute the target protein with 5-10 CV Elution Buffer. Collect the elution sample.
Resin Cleaning
After each use of the column, proteins, lipids and other substances will remain on the column. Accumulation of these residues will affect the performance of the column (capacity, fluidity, column efficiency). This is directly related to the cleanliness of the samples loaded.
It is recommended to perform a thorough column cleaning after every 2-3 uses to remove the residue on the column. There are two methods to clean the column.
Cleaning Procedure 1:
1. Wash with 5-10 CV of 30% isopropanol, ensuring 10-15 min contact time.
2. Wash with 10 CV of distilled water.
3. Equilibrate with 5-10 CV of 1X PBS with 20% ethanol before storage.
Cleaning Procedure 2:
1. Wash with 1.5M NaCl for 8-10min.
2. Wash with 10 CV of distilled water.
3. Equilibrate with 5-10 CV of 1X PBS with 20% ethanol before storage.
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