His Monoclonal Antibody

What are His-Tag monoclonal antibodies and how do they function?

His-Tag monoclonal antibodies are specialized immunoglobulins that specifically recognize and bind to polyhistidine sequences (typically 5-6 consecutive histidine residues) commonly engineered into recombinant proteins. These antibodies function by binding to histidine tags with high affinity, allowing researchers to identify, detect, and purify polyhistidine fusion proteins expressed in various systems including bacteria, insect cells, and mammalian cells .

The binding mechanism involves recognition of the histidine residues regardless of the surrounding amino acid context. Crystallographic studies have revealed that the peptide binds to a deep pocket formed at the interface of the variable domains of the light and heavy chain, primarily through hydrophobic interactions with aromatic residues and hydrogen bonds with acidic residues . The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains .

What makes His-Tag antibodies valuable in research applications?

His-Tag monoclonal antibodies offer several advantages that make them indispensable in research settings. Their high affinity (Kd = 5 × 10^-8 to 1 × 10^-9 M) enables sensitive and specific detection at low antibody concentrations (0.1-0.2 μg/ml) . This sensitivity is particularly valuable when working with low-abundance proteins or in applications requiring minimal background signal.

Additionally, these antibodies demonstrate remarkable versatility in recognizing His-tagged proteins regardless of tag position. They can detect His tags placed at N-terminal, C-terminal, and internal regions of fusion proteins . This flexibility allows researchers to design recombinant proteins with optimal tag placement for their specific experimental requirements without compromising detection capabilities.

What are the structural characteristics of His-Tag recognition?

The specificity of His-Tag monoclonal antibodies stems from their molecular structure. X-ray crystallography studies of the anti-His tag antibody 3D5 single-chain fragment (scFv) complexed with a hexahistidine peptide have revealed detailed structural insights at 2.7 Å resolution . The antibody creates a specialized binding pocket that accommodates the histidine residues through a combination of:

• Hydrophobic interactions with aromatic residues

• Hydrogen bonds with acidic residues

• Recognition of the C-terminal carboxylate group

• Interaction with the main chain of the last four residues

• Specific binding to the imidazole side-chains of the histidine residues

This structural arrangement explains why some His-Tag antibodies can recognize even partially exposed tags or those with fewer than six histidines, as seen with antibodies that can detect 4xHis and 5xHis tags .

What are the optimal protocols for His-Tag antibody-based Western blotting?

Western blotting represents one of the most common applications for His-Tag monoclonal antibodies. To achieve optimal results, researchers should consider the following methodological approach:

• Sample preparation: Prepare protein samples containing His-tagged fusion proteins using standard lysis and denaturation protocols.

• Antibody concentration: Use His-Tag antibodies at 0.1-1 μg/mL concentration depending on the expression level of the target protein .

• Blocking agent selection: For lowest background in Western blotting applications, Alkali-soluble Casein is recommended as a blocking agent .

• Secondary antibody options:

  • Goat Anti-Mouse IgG (H&L) [HRP] Polyclonal Antibody

  • MonoRab™ Anti-Mouse IgG (H&L) (76F10), mAb, Rabbit

• Signal development: Choose appropriate visualization methods based on your detection system (chemiluminescence, fluorescence, etc.).

Comparative studies have demonstrated that optimized protocols can significantly improve signal-to-noise ratios, as evidenced by Western blot experiments comparing different anti-His antibodies at equivalent concentrations (0.1 μg/mL) .

How can His-Tag antibodies be effectively used in immunoprecipitation studies?

Immunoprecipitation (IP) using His-Tag monoclonal antibodies provides a powerful approach for isolating His-tagged proteins from complex mixtures. The methodology involves:

• Preparation of cell lysates: Generate lysates from cells expressing His-tagged proteins under non-denaturing conditions to preserve native protein structure.

• Antibody immobilization: Conjugate His-Tag antibodies to protein A/G beads or other suitable matrix.

• Immunoprecipitation: Incubate the antibody-conjugated beads with cell lysates containing His-tagged fusion proteins.

• Washing and elution: Remove non-specifically bound proteins through washing steps, then elute the His-tagged proteins.

• Analysis: Typically performed by Western blotting to confirm successful isolation of the target protein .

Validation experiments should include positive controls containing known His-tagged fusion proteins and negative controls using isotype control antibodies to confirm specificity .

What considerations are important for flow cytometry applications?

Flow cytometry applications with His-Tag monoclonal antibodies require specific considerations:

• Cell preparation: For intracellular His-tagged proteins, permeabilization is necessary, while cell surface-expressed His-tagged proteins can be detected without permeabilization.

• Antibody concentration: Titration experiments are recommended to determine optimal antibody concentration (typically 1-5 μg/mL).

• Secondary detection: FITC-conjugated or other fluorophore-labeled secondary antibodies are commonly used for signal development.

• Controls: Include non-transfected cells as negative controls to establish background signal levels.

Flow cytometry data has demonstrated successful detection of His-tagged proteins in transfected CHO cells (showing positive signal) compared to non-transfected CHO cells (showing no signal) , confirming the specificity of the antibody recognition.

How does binding affinity affect experimental outcomes with His-Tag antibodies?

Binding affinity significantly impacts the sensitivity and specificity of His-Tag antibody applications. With Kd values typically ranging from 5 × 10^-8 to 1 × 10^-9 M , these antibodies demonstrate high affinity for His-tagged proteins, enabling detection at low concentrations.

Biolayer interferometry (BLI) measurements have provided quantitative insights into binding kinetics between His-Tag antibodies and various His-tagged fusion proteins. These studies reveal that:

• Position of the His-tag (N-terminal, C-terminal, or internal) can affect binding affinity

• Accessibility of the His-tag within the protein structure influences recognition efficiency

• The local environment surrounding the His-tag may modulate antibody binding

Researchers should consider these factors when designing experiments, particularly when working with complex fusion proteins where tag accessibility might be compromised.

What factors contribute to lot-to-lot variability in antibody performance?

Consistency in antibody performance across different production lots is crucial for experimental reproducibility. Analysis of lot-to-lot variability using Western blot techniques has demonstrated that high-quality His-Tag antibodies maintain consistent signal intensity across multiple batches .

Factors that can contribute to lot-to-lot variability include:

• Production conditions: Variations in cell culture conditions during antibody production

• Purification protocols: Differences in purification efficiency or methods

• Storage conditions: Improper storage leading to antibody degradation

• Quality control measures: Stringency of quality control testing between batches

To minimize the impact of lot-to-lot variability, researchers should:

• Request certificate of analysis data from manufacturers

• Perform validation tests when switching to a new lot

• Maintain consistent experimental conditions

• Consider purchasing larger quantities of a single lot for long-term projects

How can researchers optimize detection of His-tags in different structural contexts?

The accessibility of His-tags can vary significantly depending on protein structure, potentially affecting detection efficiency. Research has shown that His-Tag monoclonal antibodies can recognize tags in various structural contexts, but optimization strategies may be necessary:

• Tag position optimization: Comparative studies have demonstrated successful detection of both N-terminal and C-terminal His-tagged proteins , though accessibility may differ.

• Denaturation considerations: Some His-Tag antibodies can recognize both native and denatured forms of His-tagged proteins , but denaturation may be necessary when tags are buried within protein structures.

• Linker sequence design: Including flexible linker sequences between the protein of interest and the His-tag can improve tag accessibility.

• Tag length variations: While 6xHis is standard, some antibodies can detect 4xHis and 5xHis tags , providing flexibility when full tag exposure is limited.

Experimental data confirms that high-quality His-Tag antibodies can recognize His tags regardless of their position within the fusion protein, as demonstrated by Western blot analysis of proteins with N-terminal versus C-terminal His tags .

What strategies can address weak or absent signals in Western blot applications?

When encountering weak or absent signals when using His-Tag antibodies in Western blotting, consider the following troubleshooting approaches:

• Antibody concentration adjustment: Increase antibody concentration incrementally from 0.1 μg/mL to 1 μg/mL .

• Blocking optimization: Test different blocking agents; Alkali-soluble Casein has been recommended for lowest background .

• Exposure time modification: Increase exposure time during imaging to detect weak signals.

• Sample preparation evaluation: Ensure complete lysis and denaturation of samples containing His-tagged proteins.

• Transfer efficiency verification: Use reversible protein stains to confirm successful protein transfer to membranes.

• Secondary antibody optimization: Test alternative secondary antibodies with higher sensitivity or different conjugates.

Comparative Western blot analyses have demonstrated significant performance differences between antibodies from different sources at identical concentrations, highlighting the importance of antibody selection in achieving optimal results .

How should researchers properly reconstitute and store His-Tag monoclonal antibodies?

Proper handling, reconstitution, and storage are critical for maintaining antibody functionality:

• Reconstitution protocol:

  • Reconstitute lyophilized antibody with deionized water (or equivalent) to a final concentration of 0.5 mg/ml .

  • Some products may be lyophilized from PBS (pH 7.4) with trehalose as a protectant .

• Storage conditions:

  • Store at 2-8°C for short-term use .

  • For long-term storage, aliquot and store at -20°C or -80°C to avoid repeated freeze-thaw cycles.

• Working solution preparation:

  • Dilute to working concentration immediately before use.

  • The 100-μg package size typically provides enough purified antibody for approximately 1000 mL of working solution .

• Quality assessment:

  • Periodically validate antibody performance, especially after extended storage.

  • Antibody purity should be >95% as determined by SDS-PAGE .

Adhering to these guidelines helps ensure consistent antibody performance across experiments and maximizes the usable lifespan of the reagent.

What are the common sources of non-specific binding and how can they be mitigated?

Non-specific binding represents a significant challenge in antibody-based applications. Common sources and mitigation strategies include:

• Insufficient blocking:

  • Optimize blocking agent (Alkali-soluble Casein recommended) .

  • Increase blocking time or concentration.

• Cross-reactivity with endogenous histidine-rich proteins:

  • Include additional washing steps with increased stringency.

  • Use negative control samples lacking His-tagged proteins.

• Secondary antibody non-specific binding:

  • Include samples incubated with secondary antibody alone.

  • Consider using more specific secondary antibodies.

• Sample complexity:

  • Pre-clear complex samples with protein A/G beads before antibody application.

  • Use gradient gels for better protein separation in Western blotting.

• Antibody concentration:

  • Titrate antibody to determine optimal concentration that maximizes specific signal while minimizing background.

Comparative dot blot experiments have demonstrated variations in non-specific binding between different anti-His antibodies, highlighting the importance of antibody selection and protocol optimization for specific applications .

How can His-Tag antibodies be utilized in crystallization studies?

His-Tag antibodies offer innovative applications in protein crystallography:

The anti-His scFv crystals form 70 Å-wide channels that allow diffusion of peptides or small proteins, potentially acting as a framework for the crystallization of His-tagged target proteins . This approach provides several advantages:

• Crystallization scaffold: The antibody provides a stable framework to facilitate crystal formation of challenging proteins.

• Minimal tag requirement: The antibody can recognize very short tags of just three histidine residues, which are suitable for crystallization .

• Structural insights: Co-crystallization can provide detailed insights into the binding interface between the antibody and His-tagged proteins.

• Experimental design considerations: Researchers should optimize tag position and linker length to ensure accessibility while minimizing interference with the target protein structure.

This application represents an advanced use of His-Tag antibodies beyond traditional detection and purification purposes.

What considerations are important when using His-Tag antibodies for immobilized metal affinity chromatography (IMAC)?

While IMAC typically uses metal ions (Ni²⁺, Co²⁺, etc.) for His-tagged protein purification, antibody-based affinity purification offers complementary advantages:

• Specificity enhancement: Anti-His antibodies immobilized on a matrix can be used for affinity purification of recombinant proteins carrying very short tags (as few as three histidine residues) .

• Elution conditions: Antibody-based purification may allow for milder elution conditions compared to IMAC, potentially preserving protein activity.

• Purity considerations: Antibody-based purification can provide higher specificity than metal-based IMAC in complex samples with multiple histidine-rich proteins.

• Reusability: Properly maintained antibody columns can be reused multiple times, though capacity may decrease with repeated use.

• Scale considerations: Antibody-based purification is often more suitable for small-scale, high-purity applications rather than large-scale purification.

This approach is particularly valuable for proteins that will be subsequently used in crystallization studies or other applications where minimal tag length is desirable .

How do novel antibody formats impact His-tag detection capabilities?

Recent advances in antibody engineering have expanded the repertoire of His-Tag detection tools:

• Single-chain fragments (scFv): The crystal structure of the anti-His tag antibody 3D5 single-chain fragment has provided the basis for designing antibodies with enhanced stability and affinity .

• Chimeric antibody formats: Monoclonal Anti-His Tag Antibody, Human IgG1 (AY63) represents a chimeric antibody that combines the variable region of a mouse monoclonal antibody with a human constant domain .

• Antibody-enzyme fusions: Recombinant anti-His scFv can be fused to alkaline phosphatase to create convenient detection tools with direct enzymatic activity .

• Species variations: While many His-Tag antibodies are mouse-derived, human variants are now available that may offer advantages in certain applications .

These novel formats provide researchers with expanded options for optimizing His-tag detection based on their specific experimental requirements, potentially offering improved sensitivity, reduced background, or enhanced compatibility with particular detection systems.

What emerging applications are being developed for His-Tag monoclonal antibodies?

His-Tag monoclonal antibodies continue to evolve beyond their traditional applications in protein detection and purification. Emerging applications include:

• Structural biology tools: The use of anti-His antibodies as crystallization scaffolds represents an innovative approach to facilitate structural studies of challenging proteins .

• Single-molecule imaging: His-Tag antibodies conjugated to quantum dots or other fluorophores enable tracking of individual His-tagged proteins in real-time.

• Diagnostic applications: The high specificity of His-Tag antibodies makes them valuable tools for detecting His-tagged viral or bacterial proteins in diagnostic assays.

• Therapeutic protein analysis: His-Tag antibodies play crucial roles in characterizing recombinant therapeutic proteins during development and quality control processes.

• Biosensor development: Immobilized His-Tag antibodies can serve as capture agents in various biosensor platforms for protein detection.

As antibody engineering technologies advance, we can anticipate continued refinement of His-Tag antibodies with enhanced properties for specialized applications.

How does the integration of His-Tag antibodies with other technologies enhance research capabilities?

The integration of His-Tag antibodies with complementary technologies creates powerful research platforms:

• Multiplexed detection systems: Combining His-Tag antibodies with antibodies against other epitope tags enables simultaneous detection of multiple proteins in complex systems.

• Automation platforms: His-Tag antibody-based detection has been adapted to high-throughput screening platforms, accelerating protein interaction studies and drug discovery.

• Cryo-electron microscopy: His-Tag antibodies can facilitate protein complex formation and provide fiducial markers for cryo-EM studies.

• Affinity purification-mass spectrometry: His-Tag antibodies enable efficient isolation of protein complexes for subsequent mass spectrometry analysis and identification of interaction partners.

• Microfluidic systems: Integration with microfluidic platforms allows for rapid, low-volume analysis of His-tagged proteins for point-of-care or field applications.