IMAC Agarose (NTA) 6FF
Based on 1 Customer Validation
MCE IMAC Agarose (NTA) 6FF is prepared by covalently coupling tetradentate nitrilotriacetic acid (NTA) to an agarose matrix. It features high binding capacity, excellent specificity, and superior ligand stability. The resin allows flexible chelation with metal ions such as Zn2+, Ni2+, or Cu2+, and is suitable for the purification of His-tagged recombinant proteins expressed in bacterial, mammalian, insect, and baculovirus systems.
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Storage :
4°C, 5 years
Do not dry or freeze
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
Immobilized Metal Ion Affinity Chromatography (IMAC) is a widely used technique for the purification of proteins and peptides. It relies on the specific interactions between certain amino acid side chains—primarily histidine, and to a lesser extent, cysteine and tryptophan—and transition metal ions such as Zn2+, Ni2+, Cu2+, or Co2+. These metal ions are chelated by ligands, which are covalently attached to an insoluble polymer matrix, typically agarose.
The strength of interaction between the immobilized metal ions and a given protein or peptide depends on the type, number, and spatial distribution of relevant side chains, as well as on the nature of the metal ion itself. Chromatographic conditions, including pH, salt type and concentration, and the presence of specific additives, can also significantly influence binding affinity. In recent years, IMAC has become increasingly important for the purification of recombinant proteins carrying histidine tags, and it is now regarded as a key tool in modern protein purification workflows.
MCE IMAC Agarose (NTA) 6FF is prepared by covalently coupling tetradentate nitrilotriacetic acid (NTA) to an agarose matrix. It features high binding capacity, excellent specificity, and superior ligand stability. The resin allows flexible chelation with metal ions such as Zn2+, Ni2+, or Cu2+, and is suitable for the purification of His-tagged recombinant proteins expressed in bacterial, mammalian, insect, and baculovirus systems.
This product is a chelating resin without preloaded metal ions. The binding capacity depends on the type of metal ion used and specific experimental conditions, and should be evaluated by the user after metal ion loading.
Each 1 mL of total volume contains 0.5 mL of resin. Thoroughly resuspend the resin before use and mix well before aliquoting.
The specifications of the product correspond to the actual resin volume, with the resin content of 50%.
Protocol
Buffer Preparation
| Buffer | Composition |
|---|---|
| Binding/Wash Buffer | 50 mM Tris, 500 mM NaCl, pH 7.4 |
| Elution Buffer | 50 mM Tris, 500 mM NaCl, 200-500 mM imidazole, pH 7.4 |
| Regeneration Buffer | 50 mM Tris, 500 mM NaCl, 200 mM EDTA, pH 7.4 |
| Metal ion chelating buffer | 50 mM Tris, 200 mM metal ions (Zn2+/Ni2+/Cu2+), pH 7.4 |
| Storage Buffer | 20% Ethanol |
Note: a. It is recommended to prepare all buffers with ultrapure water. After preparation, filter them through a 0.45 μm or 0.22 μm membrane for sterilization.
b. The above buffer formulations are for reference only and can be adjusted according to experimental needs.
Protocol
It is recommended to filter the sample through a 0.22 μm or 0.45 μm membrane prior to purification.
1. Load the column: Pack IMAC Agarose (NTA) 6FF into an appropriate chromatography column. Connect the column to a chromatography system.
2. Chelation of Metal Ions: Equilibrate the chromatography column with a metal ion chelation buffer to allow chelation of the metal ions. Then wash the column with ddH2O to remove unbound metal ions.
3. Column Equilibration: Equilibrate the column with 5 column volumes (CV) of binding buffer. Repeat this step 2-3 times.
4. Sample Loading: Load the sample using a pump or sample loop. Collect the flowthrough. For improved binding efficiency, repeated sample loading is allowed.
Note: a. Select an appropriate resin volume based on the protein amount. Do not exceed the column’s binding capacity.
b. Ensure the sample solution has suitable ionic strength and pH. Dilute the sample with binding/wash buffer or dialyze against it.
c. High sample viscosity or large volumes may increase backpressure on the column.
5. Washing: Wash the column with 10-20 CV of wash buffer to remove nonspecifically bound proteins. Collect the wash fractions until the UV absorbance baseline stabilizes.
6. Elution: Elute the target protein with 5-10 CV of elution buffer. Collect elution fractions separately. The elution fractions contain the target protein.
7. Regeneration: Wash the column thoroughly with 5-10 CV of elution buffer. Re-equilibrate the column with wash buffer.
8. (Optional) Column Reuse and CIP (Cleaning-in-Place): IMAC Agarose (NTA) 6FF is reusable. However, repeated use may lead to the accumulation of impurities, causing increased backpressure or reduced capacity. Column CIP is recommended when necessary.
1) Wash the column with 5-10 CV of Regeneration Buffer to remove tightly bound nonspecific proteins, followed by 5-10 volumes of ddH2O.
2) Wash with 3-5 CV of 1 M NaOH to remove precipitates and strongly hydrophobic contaminants, followed by 5-10 volumes of ddH2O.
3) Wash with 3-5 column volumes of 70% ethanol or 30% isopropanol to remove lipid-based contaminants, followed by 5-10 CV of ddH2O.
9. Storage: After final cleaning, equilibrate the column with 5-10 CV ddH2O, followed by 5-10 CV of storage buffer. Disconnect the column and store at 4°C.
Storage
4°C, 5 years
Do not dry or freeze
Attention
1. Ensure the resin is thoroughly resuspended before use.
2. It is recommended to replace the storage buffer every 2-3 months to prevent microbial contamination due to ethanol evaporation.
3. This product is for R&D use only, not for drug, household, or other uses.
4. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Components | HY-K0255-5 mL | HY-K0255-25 mL | HY-K0255-50 mL | HY-K0255-100 mL |
|---|---|---|---|---|
| IMAC Agarose (NTA) 6FF | 5 mL | 25 mL | 50 mL | 100 mL |
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