In biopharmaceutical development, structural biology research, and functional protein analysis, efficient purification of recombinant proteins is a critical step for experimental success. The introduction of affinity tags has significantly simplified the isolation process of target proteins, among which the His tag (polyhistidine tag) is one of the most commonly used types in recombinant expression systems due to its small molecular weight, structural stability, and low immunogenicity. The His tag typically consists of 6-12 histidine residues and can be fused to the N- or C-terminus of the recombinant protein. Under specific buffer conditions, it forms stable coordination bonds with metal ions, making it ideal for selective capture and elution using immobilized metal affinity chromatography (IMAC).
The his-tagged protein purification kit offered by MtoZ Biolabs provides IMAC purification materials based on Ni²⁺-NTA and Co²⁺-IDA ligand systems. It supports multiple formats including magnetic beads, highly cross-linked agarose beads, and pre-packed columns, making it broadly applicable from high-throughput screening to pilot-scale and process development stages. This kit can help researchers efficiently isolate and purify His-tagged proteins across various experimental scenarios.
Product Overview
The his-tagged protein purification kit offers a variety of ligand-matrix combinations to accommodate diverse purification needs, from microscale screening to large-scale production. Based on differences in ligand chemistry and resin matrix, MtoZ Biolabs provides flexibility and performance tailored to specific experimental workflows. The table below summarizes the key performance parameters of each ligand-matrix combination:
|
Characteristics |
Description |
|
Product content |
25% slurry in specific protective buffer |
|
Beads size |
30~150 μm |
|
Magnetization |
Superparamagnetic |
|
Coupled Ligand |
Iminodiacetic acid (IDA) |
|
Chelating metal ion |
Ni2+ |
|
Metal ion density |
30-50 μmol/ml beads |
|
Binding capacity |
20-40 mg His-tagged fusion protein per ml beads (100%) |
|
Specificity |
Met-His-tag-Protein, His-tag-Protein, Protein-His-tag |
|
Application |
Protein purification, IP, Co-IP |
Each milliliter of Ni agarose magnetic bead suspension contains approximately 0.25 mL of packed agarose beads. The labeled volume refers to the total volume of the suspension. One milliliter of the suspension can be used to purify 5-10 mg of His-tagged protein with an approximate molecular weight of 60 kDa. The actual amount of purified target protein may vary depending on the molecular weight of the His-tagged protein.
The his-tagged protein purification kit's small-size format is sufficient for 10 purifications of His-tagged recombinant proteins, using 1-2 grams of wet bacterial pellet per purification. The maximum yield per purification is 10-15 mg, depending on the protein's molecular weight. For His-tagged recombinant proteins with a molecular weight of 60 kDa, the expected yield per run is 5-10 mg.
Protocol
The his-tagged protein purification kit is compatible with various IMAC matrix formats, including agarose beads, magnetic beads, and pre-packed chromatography columns. The following protocol is based on small-volume agarose beads; users can adjust accordingly based on the matrix used:
1. Resin Activation and Equilibration
(1) Gently pipette to resuspend the Ni-agarose magnetic bead suspension. Use 1 mL of bead suspension for every 5-10 mg of target protein (approx. 60 kDa in molecular weight). Transfer the appropriate amount of Ni-agarose beads to a clean microcentrifuge tube and remove the supernatant using magnetic separation.
(2) Add a volume of non-denaturing wash buffer equal to the original bead suspension volume to wash the beads.
(3) Gently pipette to resuspend the beads. Place the tube on a magnetic stand for 10 seconds and discard the supernatant.
(4) Repeat the washing steps twice. Use freshly washed beads.
2. His-tag Protein Purification under Native Conditions
Note: The protein sample must not contain chelating agents (e.g., EDTA, EGTA) or reducing agents (e.g., DTT, β-mercaptoethanol).
(1) Incubation with beads: Add 20 µL of well-mixed Ni-agarose bead suspension to the protein sample. Incubate at room temperature on a tilting platform or rotary mixer for 20-30 minutes.
Note: For enhanced protein stability, incubation can be performed at 2-8ºC for 1 hour with rotation.
(2) Magnetic separation: After incubation, place the tube on a magnetic stand for 10 seconds and discard the supernatant.
Note: Retain a portion of the supernatant to check the binding efficiency of the target protein.
(3) Washing: Add 100 µL of non-denaturing wash buffer, gently pipette to resuspend the beads.
(4) Place on magnetic stand for 10 seconds. Collect 20 µL of the supernatant for downstream analysis; discard the remaining. Repeat the washing step three times in total.
(5) Elution: Add 20 µL of non-denaturing elution buffer, gently invert the tube several times to resuspend the beads. Incubate for 5 minutes. Separate on a magnetic stand and collect the eluate in a new microcentrifuge tube, this is the purified His-tagged protein.
(6) If necessary, repeat the elution step once and combine the eluates in a new tube.
Features and Benefits
1. Flexible Format Options for Diverse Experimental Platforms
The product is designed to accommodate a wide range of applications, from high-throughput screening to process development.
2. High Compatibility Across Expression Systems
Suitable for use with E. coli, mammalian cells, yeast, and other expression systems. Enables one-step purification of both N- and C-terminal His-tagged proteins.
3. Good Reusability for Cost Efficiency
The high-crosslinked agarose-based matrix offers excellent chemical stability and can be reused 2-3 times, making it ideal for routine workflows or batch production.
4. Cost-Effective for Long-Term Use
Offers a favorable performance-to-price ratio, providing a practical alternative to mainstream commercial brands and helping research teams reduce project costs.
Applications
1. Purification of His-tagged Recombinant Proteins
Suitable for efficient capture of a wide range of His-tagged fusion proteins.
2. Functional and Structural Protein Studies
Provides high-purity protein samples for downstream applications such as protein-protein interaction analysis and crystallography.
3. Antigen Preparation and Immunoassay Validation
Enables the production of His-tagged antigens to improve immunogenicity and antibody quality.
4. High-throughput Screening
Compatible with automated liquid handling platforms for evaluating protein expression levels and binding efficiency.
5. Pilot-scale Process Development
Serves as a standardized purification workflow template for process optimization and scale-up trials.
For more product details or technical support, please contact MtoZ Biolabs. We are committed to providing efficient and reliable solutions for protein purification needs.
FAQs
Q1: How Should I Choose between Ni²⁺ and Co²⁺ Ligands Based on Experimental Goals?
A1: For maximum protein yield, Ni-NTA is recommended. For higher purity suitable for downstream applications, Co-IDA is preferred.
Q2: Does the Position of the His Tag Affect Protein Purification Efficiency?
A2: In theory, His tags at either the N- or C-terminus can bind effectively to IMAC resins. However, protein conformation and tag accessibility may impact binding efficiency. It's advisable to evaluate both constructs experimentally.
Q3: Is the Kit Compatible with Magnetic Bead-Based Automation Systems?
A3: Yes. The his-tagged protein purification kit includes magnetic bead formats compatible with high-throughput systems such as KingFisher. A 96-well plate version is also available for parallel screening.
Q4: Do Different Metal Resins Pose Risks of Metal Ion Leaching?
A4: Ni-NTA resins with tetradentate chelators exhibit minimal metal ion leakage. For applications sensitive to metal contamination, Co resins with additional desalting steps are recommended.
Q5: What Applications Are Magnetic Beads VS. Agarose Beads Suited for?
A5: Magnetic beads are ideal for automated high-throughput screening and rapid purification. Agarose beads are more suitable for column chromatography and routine batch purification.
Q6: Can the Resin Be Reused, and How Is Performance Affected?
A6: Highly cross-linked agarose resins can be reused 2-3 times with proper regeneration. It's recommended to regularly assess binding capacity to ensure consistent performance.
