His-Tag Monoclonal Antibody

What is a His-tag and how do His-tag monoclonal antibodies recognize it?

The His-tag is a synthetic oligo peptide consisting of 6-10 consecutive histidine residues (most commonly HHHHHH or 6×His) that can be fused to the N-terminus, C-terminus, or internal regions of recombinant proteins. His-tag monoclonal antibodies specifically recognize this histidine sequence through a combination of hydrophobic interactions with aromatic residues and hydrogen bonds with acidic residues in the antibody's binding pocket .

The crystal structure analysis of the anti-His tag antibody (3D5) shows that the antibody binds to a deep pocket formed at the interface of the variable domains of the light and heavy chains. The antibody recognizes both the C-terminal carboxylate group and the main chain of the last four residues, as well as the last three imidazole side chains of the histidine residues .

How do researchers choose between different His-tag monoclonal antibody clones?

Different His-tag monoclonal antibody clones have varying specificities and sensitivities:

When selecting a clone, researchers should consider:

• The position of the His-tag in their fusion protein

• The specific application requirements (detection method)

• The desired sensitivity and background levels

• Compatibility with other reagents in their experimental system

What advantages do His-tags offer over other epitope tags?

His-tags provide several advantages compared to other common epitope tags:

• Size: The small size of His-tags (6-10 amino acids) makes them less likely to obstruct the target protein's structure or function compared to larger epitope tags .

• Stability: His-tags remain stable under denaturing conditions, making them suitable for various experimental conditions .

• Metal Affinity: The string of histidine residues binds to several types of immobilized metal ions (nickel, cobalt, copper), enabling simple purification through metal affinity chromatography .

• Position Flexibility: His-tags can be positioned at N-terminal, C-terminal, or even internal regions of proteins while maintaining functionality .

• Minimal Impact: His-tags generally have minimal impact on protein folding, biological activity, and crystallization properties .

What are the optimal conditions for using His-tag monoclonal antibodies in Western blot applications?

For optimal Western blot detection of His-tagged proteins:

Sample Preparation:

• Use reducing conditions (for most His-tag antibodies like AD1.1.10)

• Load protein at 0.2-0.5 mg/mL concentration

Antibody Dilutions:

• Primary antibody: 1:1000-1:10000 (clone-dependent)

• Example: AD1.1.10 at 1:4000 dilution for HRP-conjugated versions

Buffer Systems:

• Immunoblot Buffer Group 1 for AD1.1.10 clone

• PBS with 0.02% sodium azide for several clones

Detection Systems:

• HRP-conjugated secondary antibodies (1:1000-1:5000)

• Fluorophore-conjugated antibodies for quantitative analysis

Membrane Type:

• PVDF membranes show superior results for His-tagged protein detection

Comparative Western blot analysis shows that using specific buffer systems and optimal antibody dilutions can significantly improve signal-to-noise ratio, as demonstrated in experiments comparing THE™ His Antibody performance against competitor antibodies at equal concentrations (0.1 μg/mL) .

How can researchers optimize His-tag detection in flow cytometry experiments?

For optimal flow cytometry detection of His-tagged proteins:

Cell Preparation:

• Fix cells with 70% ethanol (10 minutes) or Flow Cytometry Fixation Buffer

• Permeabilize with 0.25% Triton X-100 (20 minutes) for intracellular targets

• Block with 5% BSA (1 hour) at room temperature

Antibody Protocol:

• Primary antibody dilution: 1:200-1:400 in 2.5% BSA

• Incubation: 3 hours at room temperature or overnight at 4°C

• Secondary detection: Alexa Fluor 488/FITC-conjugated anti-mouse IgG

Controls:

• Include untransfected cells (negative control)

• Include isotype control antibody (e.g., MAB002)

• Use cells transfected with non-His-tagged proteins

Flow cytometry analysis of HEK293 cells transfected with His-tagged proteins shows distinct positive populations when compared to untransfected controls, with signal-to-noise ratios exceeding 10-fold when using optimized protocols with appropriate controls .

What are critical factors affecting His-tag antibody specificity and sensitivity?

Key factors that influence His-tag antibody performance include:

Epitope Accessibility:

• Protein folding can obscure internally placed His-tags

• Denaturation may be required for certain applications

• The length of linker sequences between protein and His-tag affects detection

Buffer Composition:

• pH: Optimal range 7.2-7.5 for most His-tag antibodies

• Salt concentration: 150mM NaCl is typically optimal

• Detergents: Low concentrations (0.05-0.1%) improve specificity

Antibody Characteristics:

• Affinity: KD values in the nanomolar range provide optimal detection

• Clone specificity: Some clones recognize only linear epitopes while others detect in native conditions

• Lot-to-lot consistency: Validated antibodies show consistent performance across manufacturing lots

Cross-Reactivity:

• Histidine-rich endogenous proteins can create background

• Protein A/G can interact with certain His-tag antibody isotypes

• Secondary antibody selection affects non-specific binding

Comparative dot blot analysis using three different His-tag antibodies against the same His-tagged proteins showed significant variation in sensitivity, with some antibodies detecting concentrations as low as 10 ng while others required 100+ ng for detection .

How can His-tag antibodies facilitate structural biology and protein crystallography studies?

His-tag antibodies offer several unique advantages for structural biology applications:

Co-crystallization Applications:

• Anti-His scFv crystals can serve as a framework for the crystallization of His-tagged target proteins

• The high solvent content (77% v/v) and 70Å-wide channels in antibody crystals allow diffusion of peptides or small proteins

• Antibody-assisted crystallization can facilitate structural determination of difficult-to-crystallize proteins

Minimal Tag Requirements:

• Purification can be achieved with as few as three histidine residues when using specialized anti-His antibodies

• Shorter tags are advantageous for crystallization purposes, reducing tag-induced artifacts

Direct Structure Determination:

• The crystal structure of anti-His scFv in complex with hexahistidine peptide provides a molecular framework for understanding antibody-epitope interactions

• This structural information enables rational design of antibodies with enhanced stability and affinity

Researchers have successfully used anti-His antibodies to stabilize flexible regions of target proteins, improving diffraction quality and facilitating structure determination of otherwise challenging protein targets .

How should researchers troubleshoot weak or non-specific signals in His-tag detection experiments?

When encountering detection problems with His-tagged proteins, follow this systematic troubleshooting approach:

Problem: Weak or No Signal

Problem: High Background/Non-specific Bands

Evidence from comparative Western blot experiments shows that optimizing antibody concentration (0.1-1.0 μg/mL) and properly validating experimental conditions with appropriate controls significantly improves signal-to-noise ratio and eliminates non-specific bands .

What considerations are important when using His-tag antibodies for immunoprecipitation (IP) studies?

For successful immunoprecipitation of His-tagged proteins:

Antibody Selection:

• Optimal dilution range: 1:100-1:500 for IP applications

• Preferred clones: HIS.H8 and 4F3 show superior IP performance

• Consider using HRP-conjugated versions to eliminate secondary detection steps

Protocol Optimization:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use mild lysis conditions to preserve protein-protein interactions

• Include appropriate detergents (0.1-0.5% NP-40 or Triton X-100) to reduce background

• Perform stringent washing steps (at least 3-5 washes) with decreasing salt concentration

Validation Controls:

• Include isotype control antibody (e.g., A01007) for comparison

• Use non-tagged protein expression as negative control

• Include input sample (5-10%) to verify IP efficiency

Immunoprecipitation experiments from cell lysates containing His-tagged fusion proteins show that proper antibody selection and protocol optimization can achieve >90% pulldown efficiency with minimal non-specific binding, as demonstrated by comparative analysis between specific anti-His antibodies and isotype controls .

How are His-tag antibodies advancing protein-protein interaction studies?

His-tag antibodies enable several sophisticated approaches for studying protein-protein interactions:

Pull-down Competition Assays:

• Demonstrate competitive binding between proteins by using differentially tagged constructs

• Example: Mixtures of HIS-tagged and FLAG-tagged proteins competing for binding to GST-tagged targets can be analyzed using anti-HIS antibodies

• Quantify interaction affinities by measuring displacement with increasing concentrations of competitor proteins

Direct Binding Assays:

• Investigate direct protein-protein interactions using His-tagged proteins

• Example: GST-Shank3 directly binding to His-tagged STIM1 was confirmed using anti-His antibodies in pull-down assays

• Establish interaction domains by testing binding with truncated constructs

Multi-protein Complex Analysis:

• Characterize protein complexes by sequential immunoprecipitation using different tag antibodies

• Example: His-tag antibodies identified direct binding between Pit-1 protein and cryptic ESRRG splice variants

These methodologies provide researchers with tools to dissect complex biomolecular interactions with high specificity and sensitivity, enabling the characterization of previously challenging protein-protein interaction networks.

What recent innovations have improved His-tag antibody performance in challenging applications?

Several technological advances have enhanced His-tag antibody utility:

Enhanced Sensitivity and Specificity:

• Next-generation His-tag antibodies demonstrate lot-to-lot consistency with standardized performance metrics

• Comparative analysis shows up to 10-fold improvements in sensitivity between older and newer generation antibodies

Application-Specific Modifications:

• Direct conjugation to detection enzymes (HRP) eliminates secondary antibody requirements and reduces background

• Fluorophore-conjugated versions enable multiplexed detection with other epitope tags

• Single-chain fragment (scFv) derivatives offer advantages for structural applications and improved tissue penetration

Cross-Platform Validation:

• Modern His-tag antibodies undergo rigorous validation across multiple detection methods

• Example: THE™ His Antibody demonstrated consistent performance across Western blot, dot blot, flow cytometry, and immunoprecipitation applications

Biolayer interferometry (BLI) measurements of binding affinity between THE™ His Tag Antibody and different His-tagged fusion proteins confirm that newer antibody generations maintain consistent recognition regardless of tag position within the target protein .

How can researchers determine the optimal His-tag antibody concentration for different experimental systems?

Determining optimal antibody concentration requires systematic titration across applications:

Western Blot Optimization:

• Starting dilution range: 1:1000-1:10000

• Perform serial dilutions (1:1000, 1:2000, 1:5000, 1:10000)

• Evaluate signal-to-noise ratio at each concentration

• Select the highest dilution that maintains robust signal with minimal background

Flow Cytometry Titration:

• Starting concentration: 1-10 μg/mL

• Test 2-fold serial dilutions

• Calculate staining index (median positive/median negative) for each concentration

• Plot titration curve to identify saturation point

Immunofluorescence Optimization:

• Initial dilution: 1:50-1:200

• Assess background in negative controls at each concentration

• Select dilution that maximizes specific signal while minimizing non-specific staining

ELISA Calibration:

• Generate standard curves with known concentrations of His-tagged proteins

• Test antibody dilutions from 1:500-1:10000

• Calculate detection limits and linear range for each concentration

• Select optimal dilution based on assay sensitivity requirements

Experimental data from Western blot analysis shows that while some applications require higher antibody concentrations (1 μg/mL), others achieve optimal results at significantly lower concentrations (0.1 μg/mL), highlighting the importance of systematic titration for each experimental system .

How should researchers interpret variations in band patterns when detecting His-tagged proteins?

Band pattern variations require careful interpretation:

Multiple Bands Analysis:

Quantitative Considerations:

• Evaluate band intensity using densitometry software

• Compare relative expression levels between samples

• Consider loading controls for accurate quantification

Scientific data from Western blot experiments shows that His-tagged fusion proteins may appear at different molecular weights than predicted (32 kDa, 65 kDa, and 120 kDa bands have been observed for proteins with expected sizes of 28 kDa, 60 kDa, and 115 kDa, respectively), likely due to effects of the His-tag on protein migration .

What experimental controls are essential for validating His-tag antibody specificity?

A comprehensive control strategy includes:

Positive Controls:

• Purified His-tagged recombinant protein

• Lysate from cells expressing known His-tagged protein

• Multiple Tag Cell Lysate containing tagged proteins

Negative Controls:

• Non-transfected cell lysates

• Lysates from cells expressing non-His-tagged proteins

• Lysates from cells expressing different epitope tags

Antibody Controls:

• Isotype control antibody (same immunoglobulin class)

• Secondary antibody only (omit primary antibody)

• Competitive inhibition with free His-peptide

Protocol Controls:

• Varying protein concentrations to assess linearity

• Different exposure times to avoid saturation

• Multiple antibody dilutions to determine specificity range

Experimental validation using flow cytometry demonstrates the importance of proper controls: comparing CHO cells transfected with His-tagged protein (positive signal) against non-transfected CHO cells (negative control) clearly established specificity parameters for THE™ His Tag Antibody detection .