Ni-IDA Magnetic Agarose Beads
Based on 1 Customer Validation
MCE Ni-IDA Magnetic Agarose Beads can be used for the detection and purification of His-tag proteins from various expression sources.
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Storage :
4℃, 2 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
MCE Ni-IDA Magnetic Agarose Beads is a highly cross-linked 4% magnetic agarose, 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. It has high loading capacity, exceptional specificity, and stability, can be used for the detection and 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 beads volume, with the beads 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.
Magnetic Bead Purification
1. Bead Pretreatment: Thoroughly resuspend the Ni-IDA Magnetic Agarose Beads, transfer the required volume into a tube, perform magnetic separation, discard the storage solution, and wash with 5 bead volumes of Lysis Buffer 2-3 times.
2. Sample Binding: Add the clarified sample and incubate on a rotator at 4°C for 1-2 h or overnight. Perform magnetic separation and collect the flow-through.
3. Washing: Wash with 5-10 bead volumes of Wash Buffer 3-5 times. Optimize the imidazole concentration according to target-protein binding and contaminant removal.
4. Elution: Add 3-5 bead volumes of Elution Buffer, incubate at room temperature for 5-10 min, perform magnetic separation, and collect the supernatant. Repeat 2-3 times if necessary. Use the corresponding urea-containing buffers for denaturing purification.
5. SDS-PAGE Analysis: Analyze the original sample, flow-through, wash, and elution fractions.
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-K0240-1 mL | HY-K0240-5 mL | HY-K0240-10 mL |
|---|---|---|---|
| Ni-IDA Magnetic Agarose Beads | 1 mL | 5 mL | 10 mL |
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