TTC14 Antibody, HRP conjugated

What is TTC14 protein and what antibodies are available for its detection?

TTC14 (Tetratricopeptide repeat protein 14) is also known by alternative names including KIAA1980, UNQ5813/PRO19630, and TPR repeat protein 14. Commercial antibodies such as the rabbit recombinant monoclonal TTC14 antibody are available for research applications. These antibodies have been validated for detecting TTC14 in human samples with a predicted band size of approximately 88 kDa . The tetratricopeptide repeat domain structure is characteristic of this protein, which is part of a broader family of proteins containing TPR motifs that often function in protein-protein interactions.

What applications are suitable for TTC14 antibodies?

Based on validated testing, TTC14 antibodies have demonstrated effectiveness in several experimental applications including:

• Western blotting (WB): Successfully detects TTC14 in human cell lines including Daudi, Ramos, and Jurkat cell lysates at dilutions of 1/1000

• Flow cytometry (intracellular): Effective at 1/100 dilution for detecting intracellular TTC14 in permeabilized Jurkat cells

The antibody's specificity for human samples has been thoroughly tested, though applications with other species may be possible based on sequence homology predictions .

What is HRP conjugation and why is it used with antibodies?

Horseradish peroxidase (HRP) conjugation involves the chemical coupling of HRP enzyme molecules to antibodies to create detection reagents for immunoassays. HRP is a heme glycoprotein of approximately 44 kDa containing about 18% carbohydrate content surrounding a protein core . HRP is preferred for antibody labeling in research applications for several key reasons:

• As a plant protein, it lacks potentially interfering autoantibodies in biological samples

• It demonstrates excellent structural stability

• It offers high enzymatic activity with various substrates

• Its carbohydrate moieties facilitate chemical conjugation strategies

The resulting HRP-antibody conjugates serve as powerful tools in immunological detection methods, particularly ELISA and Western blotting, where the enzymatic activity of HRP generates detectable signals when appropriate substrates are added.

What are the primary methods for conjugating HRP to antibodies?

Several chemical approaches exist for conjugating HRP to antibodies, each with distinct mechanisms:

• Periodate method: The most commonly used approach that oxidizes carbohydrate moieties on HRP to generate aldehyde groups that can react with amino groups on antibodies

• Glutaraldehyde method: Uses glutaraldehyde as a homobifunctional crosslinker between amino groups on both the enzyme and antibody

• Maleimide conjugation: Targets sulfhydryl groups on reduced antibodies

• EDC (1-ethyl-3-[3-dimethylaminopropyl]) method: Creates amide bonds between carboxyl groups and amino groups

The choice of method significantly impacts conjugate performance, with the periodate method being particularly advantageous for HRP due to its glycoprotein nature, allowing modification of carbohydrate moieties rather than the protein core that contains the catalytic site .

How does the classical periodate method for HRP conjugation work?

The classical periodate method follows this general procedure:

• HRP activation: Oxidation of carbohydrate moieties on HRP using sodium metaperiodate (typically 0.15M) to generate reactive aldehyde groups

• Purification: Removal of excess periodate through dialysis against phosphate buffered saline (PBS)

• Conjugation: Mixing of activated HRP with antibody (typically at defined molar ratios)

• Schiff's base formation: Aldehydes on the activated HRP react with amino groups on the antibody to form Schiff's bases

• Reduction: Addition of sodium cyanoborohydride to stabilize the conjugate by reducing the Schiff's bases to stable secondary amines

• Purification: Final dialysis against PBS to remove unreacted components

This method preserves both the antigen-binding capacity of the antibody and the enzymatic activity of HRP, making it suitable for producing functional immunodetection reagents.

How can lyophilization enhance HRP-antibody conjugation efficiency?

An enhanced conjugation protocol incorporating lyophilization has demonstrated significant improvements in conjugate sensitivity. The modified procedure involves:

• Standard HRP activation with sodium metaperiodate (0.15M)

• Dialysis of activated HRP against PBS

• Freezing of the activated HRP at -80°C for 5-6 hours

• Lyophilization (freeze-drying) of the frozen activated HRP overnight

• Mixing lyophilized activated HRP with antibody (1:4 molar ratio of antibody to HRP)

• Incubation at 37°C for 1 hour

• Addition of sodium cyanoborohydride for Schiff's base reduction

• Final dialysis against PBS

This lyophilization step fundamentally enhances the conjugation efficiency by:

• Concentrating the reactants without changing their amounts

• Reducing the reaction volume, thereby increasing the collision frequency between antibody and activated HRP molecules

• Enabling more HRP molecules to bind per antibody, creating a poly-HRP effect

Comparative testing showed that conjugates prepared using this modified method achieved functional detection at dilutions as high as 1:5000, compared to only 1:25 for conjugates prepared by the classical method - representing a 200-fold improvement in sensitivity (p<0.001) .

How should researchers optimize dilutions for HRP-conjugated antibodies?

Optimization of HRP-conjugated antibody dilutions requires systematic titration experiments with consideration of:

• Conjugation method: Antibodies conjugated using enhanced methods (like lyophilization-assisted conjugation) typically require much higher dilutions than those produced by classical methods

• Signal-to-noise ratio: The optimal dilution provides maximum specific signal with minimal background

• Antigen concentration: Lower antigen concentrations require more sensitive detection and thus may benefit from conjugates with higher HRP loading

Research data demonstrates that conjugates prepared using the lyophilization-enhanced method can detect antigens at concentrations as low as 1.5 ng and function effectively at dilutions of 1:5000, while classically prepared conjugates require much lower dilutions (1:25) for equivalent detection .

What validation should be performed for HRP-conjugated TTC14 antibodies?

Comprehensive validation of HRP-conjugated TTC14 antibodies should include:

• Spectrophotometric analysis: Wavelength scans (280-800 nm) to confirm successful conjugation, with expected peaks for antibody (280 nm) and a modified peak for HRP (around 430 nm)

• SDS-PAGE analysis: Comparing migration patterns of conjugates (under reducing and non-reducing conditions) with unconjugated antibody and HRP to confirm increased molecular weight

• Functional validation: Direct ELISA testing with target antigen to assess:

  • Detection sensitivity

  • Working dilution range

  • Signal linearity

  • Specificity for TTC14 versus related proteins

• Cell line validation: Testing with known TTC14-expressing cell lines (e.g., Jurkat, Daudi, Ramos) and negative controls to confirm specific detection patterns

What commercial solutions exist for rapid HRP conjugation to TTC14 antibodies?

Several commercial systems have been developed to simplify and standardize the HRP conjugation process:

The LYNX Rapid HRP Antibody Conjugation Kit offers a streamlined approach using pre-prepared lyophilized mixtures containing HRP. This system enables:

• Direct conjugation at near-neutral pH

• High conjugation efficiency with 100% antibody recovery

• Directional covalent bonding of HRP to the antibody

• Flexibility in conjugating small quantities of antibody/protein

• A simplified workflow with proprietary activation reagents

These kits contain lyophilized HRP mix, modifier reagent, and quencher reagent, available in various scales from 10 μg to 5 mg to accommodate different research needs .

How do rapid conjugation technologies differ from traditional methods?

Rapid conjugation technologies offer several advantages over traditional periodate methods:

These rapid technologies are particularly valuable when working with limited amounts of valuable antibodies such as those targeting specialized proteins like TTC14 .

What are the critical factors affecting stability of HRP-conjugated antibodies?

Several factors significantly impact the stability and shelf-life of HRP-conjugated antibodies:

• Storage temperature: Long-term storage at -20°C, with working aliquots at 4°C

• Protein concentration: Higher concentrations generally improve stability

• Stabilizing additives: Common stabilizers include:

  • BSA (0.1-1%)

  • Glycerol (25-50%)

  • Thimerosal or sodium azide as preservatives (Note: sodium azide can inhibit HRP at high concentrations)

• Freeze-thaw cycles: Multiple cycles significantly reduce activity

• Light exposure: HRP is light-sensitive and should be stored in amber vials or protected from light

For enhanced stability of TTC14 antibody-HRP conjugates, commercial stabilizers can be added immediately after conjugation preparation .

How does HRP detection compare with other enzyme conjugation methods?

Comparative analysis of enzyme conjugation systems for antibody labeling:

HRP remains the most widely used enzyme conjugate for TTC14 and other antibodies due to its excellent combination of small size, high activity, stability, and cost-effectiveness . This makes it particularly suitable for applications requiring high sensitivity such as detecting potentially low-abundance proteins like TTC14.

What methodological approaches can enhance detection sensitivity with HRP-conjugated TTC14 antibodies?

Several methodological refinements can significantly improve detection sensitivity:

• Poly-HRP conjugation: The lyophilization-enhanced conjugation method enables more HRP molecules to bind per antibody, creating a poly-HRP effect that significantly increases signal amplification

• Substrate selection:

  • TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection

  • Enhanced chemiluminescent (ECL) substrates for higher sensitivity

  • Tyramide signal amplification (TSA) systems for ultra-sensitive detection

• Signal development optimization:

  • Temperature control during development

  • Extended substrate incubation times

  • Optimal pH conditions

• Sample preparation optimization:

  • Effective blocking to reduce background

  • Careful antibody titration to determine optimal signal-to-noise ratio

Experimental data demonstrates that poly-HRP conjugates prepared using the lyophilization-enhanced method can detect antigens at concentrations as low as 1.5 ng, offering detection levels suitable for low-abundance targets .