Ni-IDA Agarose
MCE Ni-IDA Agarose can be used for the detection and purification of His-tag proteins.
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Storage :
4℃, 2 years
Do not dry or freeze
- Manual (2855KB)
- COA
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
MCE Ni-IDA Agarose is a highly cross-linked 4% agarose gel, synthesized through the chemically directed, high-density attachment of iminodiacetic acid (IDA). When chelated with Ni2+, it forms an extremely stable planar tetragonal structure, providing three ionic bonding sites. This configuration allows for the higher affinity purification of His-tag proteins from various expression sources such as E.coli, yeast, insect cells, and mammalian cells.
The specifications of the product correspond to the actual resin volume, with the resin content of 50%.
Protocol
Recommended Buffers (Not Provided)
Buffers for Soluble His-Tagged Proteins
| Buffer | Composition |
|---|---|
| Lysis Buffer | 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 |
| Wash Buffer | 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0 |
| Elution Buffer | 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0 |
Buffers for His-Tagged Inclusion Body Proteins
| Buffer | Composition |
|---|---|
| Denaturing Lysis Buffer | 8 M urea, 100 mM NaH2PO4, 100 mM Tris-HCl, pH 8.0 |
| Denaturing Wash Buffer | 8 M urea, 100 mM NaH2PO4, 100 mM Tris-HCl, pH 6.3 |
| Denaturing Elution Buffer | 8 M urea, 100 mM NaH2PO4, 100 mM Tris-HCl, pH 4.5 |
Note: Filter all buffers through a 0.22 μm or 0.45 μm membrane before use. Avoid EDTA, EGTA, and high concentrations of DTT, β-mercaptoethanol, or other substances that may interfere with metal-ion binding.
Sample Preparation
Intracellular proteins expressed in bacteria or yeast: Collect the cell pellet, resuspend it in Lysis Buffer at approximately 1:10 (W/V), add a suitable protease inhibitor, and disrupt the cells on ice. RNase A and DNase I may be added to reduce viscosity. Centrifuge at high speed at 4°C, collect the supernatant, and filter it through a 0.22 μm or 0.45 μm membrane.
Secreted proteins: Clarify the culture supernatant. If EDTA, histidine, or reducing agents are present, dialyze against Lysis Buffer before purification. Large-volume supernatants may be concentrated first.
Inclusion body proteins: Wash the inclusion body pellet with Lysis Buffer without urea, then resuspend it in Denaturing Lysis Buffer containing 8 M urea and purify under denaturing conditions.
Column Purification
1. Column Packing and Equilibration: Pack the Ni-IDA Agarose into a suitable column and equilibrate with at least 5 column volumes of Lysis Buffer 2-3 times, until the UV baseline is stable.
2. Sample Loading: Load using a pump, sample loop, or gravity flow and collect the flow-through. Do not exceed the binding capacity. High sample viscosity or volume may increase back pressure.
3. Washing: Wash with 10-20 column volumes of Wash Buffer until the OD280 or UV absorbance reaches a stable baseline.
4. Elution: Elute with 5-10 column volumes of Elution Buffer and collect the target protein in fractions. Use the corresponding urea-containing buffers for denaturing purification.
5. Sample Processing: Remove imidazole, urea, and salts by dialysis or desalting as required, and assess purification by SDS-PAGE.
Resin Regeneration
When the resin color becomes lighter or the binding capacity decreases, sequentially wash with 2 column volumes of 0.2 M acetic acid containing 6 M GuHCl, 5 column volumes of deionized water, 3 column volumes of 2% SDS, 5 column volumes of deionized water, 5 column volumes of absolute ethanol, 5 column volumes of deionized water, 5 column volumes of 100 mM EDTA (pH 8.0), 5 column volumes of deionized water, 5 column volumes of 100 mM NiSO4, and 10 column volumes of deionized water.
The regenerated resin may be used immediately or equilibrated with storage buffer and stored at 2-8°C. Do not freeze or dry.
Storage
4℃, 2 years
Do not dry or freeze
Components
| Components | HY-K0238-5 mL | HY-K0238-10 mL | HY-K0238-50 mL |
|---|---|---|---|
| Ni-IDA Agarose | 5 mL | 10 mL | 10 mL × 5 |
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