PAE12 Antibody

What is PAE12 Antibody and what is its target antigen?

PAE12 Antibody appears to be related to a polyclonal antibody raised against Ephrin B2 (EFNB2). It is a rabbit polyclonal antibody that has been specifically selected for its ability to recognize EFNB2 in immunohistochemical staining and western blotting applications. The target antigen EFNB2 is also known by several alternative names including EPLG5, HTKL, Htk-L, LERK5, HTK Ligand, and Eph-Related Receptor Tyrosine Kinase Ligand 5 .

The antibody is purified through a two-step process involving antigen-specific affinity chromatography followed by Protein A affinity chromatography, ensuring high specificity for its target . The polyclonal nature of this antibody means it recognizes multiple epitopes on the target antigen, providing robust detection capabilities across various experimental conditions.

What are the recommended applications and working dilutions for PAE12 Antibody?

PAE12 Antibody can be used in multiple research applications with specific working dilutions:

It's important to note that these are general guidelines, and as emphasized in product documentation, "optimal working dilutions must be determined by end user" . For each new experimental system or sample type, a titration experiment should be performed to determine the optimal antibody concentration that provides the highest signal-to-noise ratio.

How should PAE12 Antibody be stored and handled for optimal performance?

For optimal performance and longevity, PAE12 Antibody should be stored according to these guidelines:

• For frequent use: Store at 4°C (refrigeration)

• For long-term storage: Store at -20°C in a manual defrost freezer for up to two years without detectable loss of activity

• Avoid repeated freeze-thaw cycles as these can degrade antibody performance

The antibody is typically formulated in PBS (pH 7.4) containing preservatives like 0.02% sodium azide (NaN₃) and stabilizers such as 50% glycerol . This formulation helps maintain antibody stability during storage and use.

The thermal stability of antibodies like PAE12 can be assessed through accelerated thermal degradation tests. When incubated at 37°C for 48 hours, properly stored antibodies should show no obvious degradation or precipitation, with a loss rate of less than 5% within the expiration date under appropriate storage conditions .

What is the difference between polyclonal and monoclonal antibodies in research applications?

Understanding the fundamental differences between polyclonal and monoclonal antibodies is crucial for selecting the appropriate reagent for specific research applications:

Polyclonal Antibodies (like PAE12):

• Produced by multiple B-cell clones in response to an immunogen

• Recognize multiple epitopes on the target antigen

• Can be produced relatively quickly (in several months) with lower cost

• Stable over a broad range of pH and salt concentrations

• Superior for detecting denatured proteins and for immunoprecipitation of complex antigens

• Generated by numerous B-cell clones each producing antibodies to a specific epitope

• Particularly useful when the target protein undergoes conformational changes or post-translational modifications

Monoclonal Antibodies:

• Produced by a single B-cell clone

• Recognize only one specific epitope on the target antigen

• Result in homogeneous, mono-specific antibodies

• Interact with a single specific surface antigen, activating B lymphocytes to divide and differentiate into plasma cell clones that further recruit homogeneous antibodies

• Offer higher consistency between batches

• More suitable for therapeutic applications requiring high specificity

The choice between polyclonal and monoclonal antibodies depends on the specific research application, with polyclonal antibodies like PAE12 offering broader epitope recognition at the cost of potential increased background compared to monoclonal antibodies.

What controls should be implemented when working with PAE12 Antibody?

Implementing appropriate controls is essential for ensuring the validity and reliability of results when working with PAE12 Antibody:

Primary Controls:

• Positive Control: Include samples known to express the target protein (EFNB2) to confirm detection capability

• Negative Control: Include samples known not to express the target protein to assess non-specific binding

• Isotype Control: Use a non-specific antibody of the same isotype (IgG for PAE12) to evaluate background binding

Additional Validation Controls:
4. Peptide Competition/Neutralization: If available, use the immunizing peptide to neutralize the antibody and confirm specificity
5. Secondary Antibody-Only Control: Omit the primary antibody to check for non-specific binding of the secondary antibody
6. Multiple Detection Methods: Validate findings using different techniques (WB, IHC, etc.)

Application-Specific Controls:
7. For Western Blot: Include molecular weight markers to confirm target protein size
8. For IHC/ICC: Include tissue/cell sections known to differentially express the target protein
9. For Flow Cytometry: Include fluorescence-minus-one (FMO) controls

These controls help distinguish between specific signals and experimental artifacts, ensuring the reliability of results obtained with PAE12 Antibody.

What mechanisms explain cross-reactivity of PAE12 Antibody with related proteins?

Cross-reactivity of antibodies with related proteins occurs through several molecular mechanisms that are important to understand for accurate data interpretation:

Epitope Similarity Mechanisms:

• Shared Conserved Domains: Cross-reactivity often occurs when different proteins share structurally similar domains with conserved amino acid sequences

• Critical Position Homology: As demonstrated in studies with RAP proteins, antibodies can recognize epitopes with "shared homologous amino acids in critical positions" even when there are no contiguous sequence similarities

• Conformational Mimicry: Three-dimensional structural similarity between epitopes can lead to cross-recognition even without primary sequence similarity

Species Cross-Reactivity Factors:
PAE12 Antibody may exhibit cross-reactivity across species (human, mouse, pig) due to evolutionary conservation of the target epitope . This conservation reflects the biological importance of maintained protein structure and function across species.

Experimental Factors Affecting Cross-Reactivity:

• Protein Conformation: Reduction state significantly impacts epitope accessibility and recognition. In some cases, antibodies strongly recognize reduced forms of proteins but fail to recognize native forms due to disulfide bridges affecting epitope accessibility

• Antibody Concentration: Higher concentrations can increase cross-reactivity with structurally similar epitopes

• Buffer Conditions: Salt concentration, pH, and detergents can alter protein conformation and epitope exposure

Understanding these mechanisms can help researchers anticipate and distinguish between specific signal and cross-reactive background when using PAE12 Antibody in experimental systems with multiple related proteins.

How can peptide aptamer screening approaches utilize PAE12 Antibody?

Peptide aptamer screening is a powerful approach for identifying specific protein-protein interactions, and PAE12 Antibody can be effectively integrated into this methodology:

Methodological Approach for Screening:

• Immobilization of PAE12:

  • Coat plates with 20 μg of PAE12 Antibody diluted in 1 mL of sterile water

  • Incubate for 1 hour at room temperature (20-25°C)

  • Wash thoroughly with sterile water

• Blocking Non-specific Binding Sites:

  • Apply blocking solution (1% non-fat dried milk, 150 mM NaCl, 1% α-methyl mannoside)

  • Incubate with gentle agitation for 1 hour

  • This minimizes background and increases specificity of detected interactions

• Peptide Library Application:

  • Add induced peptide aptamer library to the plates

  • Gently agitate at 75 rpm for 1 minute

  • Incubate for 1 hour at room temperature

• Detection and Analysis:

  • Employ Western blotting with appropriate detection antibodies

  • Use HRP-conjugated secondary antibodies and chemiluminescent substrates for visualization

  • Analyze binding patterns to identify specific interactions

Applications of This Approach:

This methodology can be particularly valuable for:

• Identifying peptide aptamers that mimic the natural epitopes recognized by PAE12

• Developing mimotopes for diagnostic applications

• Studying the structural requirements for PAE12 binding

• Creating potential therapeutic peptides that could modulate the target protein's activity

Peptide aptamers isolated through this screening can serve as "miniscule peptides that mimic linear, intermittent, or non-peptide epitopes" of the target antigen, providing valuable tools for both basic research and applied diagnostic development.

What technical challenges arise when using PAE12 Antibody for detecting low-abundance proteins?

Detection of low-abundance proteins presents several technical challenges that require specific methodological adaptations:

Major Technical Challenges:

• Signal-to-Noise Limitations:

  • Background noise can mask signals from low-abundance targets

  • Polyclonal antibodies may exhibit higher background than monoclonal antibodies

  • Solution: Implement pre-absorption steps to deplete non-specific binders

• Detection Threshold Constraints:

  • Standard detection methods may lack sufficient sensitivity

  • Western blot chemiluminescence typically requires ~0.1 ng of protein for detection

  • Solution: Employ signal amplification techniques such as tyramide signal amplification

• Sample Processing Losses:

  • Each sample processing step can result in protein loss

  • Low-abundance proteins are disproportionately affected

  • Solution: Minimize sample handling steps and optimize protein extraction

Methodological Solutions Table:

Advanced Approaches:

• Consider upstream enrichment through immunoprecipitation before detection

• Implement computational analysis of binding profiles to enhance sensitivity

• Use high-throughput sequencing and computational modeling to detect binding patterns even with minimal signal

These methodological adaptations can significantly improve the detection of low-abundance proteins when working with PAE12 Antibody.

How can computational antibody design approaches optimize PAE12 Antibody specificity?

Computational approaches offer powerful tools for optimizing antibody specificity through rational design:

Computational Optimization Workflow:

• Structural Modeling:

  • Predict antibody structure using homology modeling with de novo CDR loop conformation prediction

  • Perform batch modeling to rapidly analyze structural variants

  • This establishes the structural basis for understanding antigen recognition

• Binding Mode Analysis:

  • Identify distinct binding modes associated with specific ligands

  • Apply energy functions (E) to characterize each binding mode (w)

  • This disentangles complex binding profiles even for chemically similar targets

• Specificity Engineering:

  • For enhanced specificity: Minimize energy functions for desired ligands while maximizing for undesired ligands

  • For cross-reactivity: Jointly minimize energy functions for all desired targets

  • This computational approach allows customization of binding profiles

Advanced Computational Tools:

Experimental Validation:
After computational design, variants must be experimentally validated. Studies have demonstrated successful "computational design of antibodies with customized specificity profiles, either with specific high affinity for a particular target ligand, or with cross-specificity for multiple target ligands" .

This computational-experimental pipeline represents a sophisticated approach to antibody engineering that can significantly enhance PAE12 Antibody specificity for challenging research applications.

What factors influence epitope recognition by PAE12 Antibody under different experimental conditions?

Epitope recognition is influenced by multiple factors that researchers should consider when designing experiments:

Critical Factors Affecting Epitope Recognition:

• Protein Conformation:

  • Reduction state dramatically affects epitope accessibility

  • Studies show antibodies can strongly recognize reduced forms of proteins but fail with non-reduced forms

  • This occurs because disulfide bridges adjacent to epitopes can alter accessibility

• Sample Preparation Impact:

  • Different preparation methods expose different epitopes

  • Denaturation can expose internal epitopes normally hidden in native proteins

  • Example: In some studies, antibodies recognized proteins only after denaturation with SDS and heat

• Buffer and Environmental Conditions:

• Fixation Methods (for IHC/ICC):

  • Paraformaldehyde preserves structure but can mask epitopes

  • Methanol/acetone better preserves some epitopes but disrupts membranes

  • Antigen retrieval methods can recover masked epitopes

• Blocking Reagents:

  • Different blocking agents (BSA, milk, serum) can differentially affect epitope accessibility

  • Non-fat dried milk (5% in TBS) is commonly used but may contain bioactive compounds

Experimental Evidence:
Research has demonstrated that epitope recognition can be dramatically affected by experimental conditions. In one study, antibodies raised against an octapeptide recognized the reduced form of a target protein strongly but failed to recognize or only weakly recognized the non-reduced form due to a disulfide bridge adjacent to the epitope .

Understanding these factors allows researchers to optimize experimental conditions for consistent and specific epitope recognition with PAE12 Antibody.

What are the optimal dilution ratios for using PAE12 Antibody in different applications?

Determining optimal dilution ratios is critical for balancing specific signal detection and minimizing background:

Recommended Dilution Ranges by Application:

Methodological Approach to Dilution Optimization:

• Pilot Titration Experiment:

  • Prepare a dilution series (e.g., 1:100, 1:500, 1:1000, 1:5000)

  • Test on known positive and negative samples

  • Analyze signal-to-noise ratio at each dilution

• Analysis Factors:

  • Specific signal intensity

  • Background levels

  • Signal-to-noise ratio

  • Consistency across replicates

• Calculation for Known Concentrations:

  • If antibody concentration is 0.5mg/mL (as for similar antibodies) :

    • For 1μg/mL working concentration: Dilute 1:500

    • For 10μg/mL working concentration: Dilute 1:50

Application-Specific Considerations:

• Western blotting often requires lower concentrations than immunostaining techniques

• Fresh tissue samples may require different dilutions than fixed samples

• Signal amplification systems can allow for higher dilutions

• Target protein abundance varies across tissues/cells, requiring application-specific optimization

As emphasized in product documentation, "optimal working dilutions must be determined by end user" through systematic testing in your specific experimental system.

How can PAE12 Antibody be validated for specificity in experimental settings?

Comprehensive validation ensures reliable experimental results and should include multiple complementary approaches:

Multi-Method Validation Strategy:

• Western Blot Analysis:

  • Run samples known to express or not express the target protein

  • Confirm band at expected molecular weight (e.g., 35-kDa for target protein)

  • Look for absence of bands in negative control samples

  • Gradient gels can provide better resolution for closely related proteins

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Should eliminate or significantly reduce specific signal

  • Maintain same concentration of antibody in both competed and non-competed samples

  • Immunizing peptides are often available specifically for neutralization experiments

• Knockout/Knockdown Validation:

  • Test on samples with genetic knockout or siRNA knockdown of target

  • Signal should be absent or significantly reduced compared to wild-type

  • Include rescue experiments to confirm specificity

• Multiple Antibody Validation:

  • Use antibodies targeting different epitopes of the same protein

  • Compare staining/binding patterns across antibodies

  • Concordant results from different antibodies increase confidence in specificity

• Mass Spectrometry Confirmation:

  • Immunoprecipitate with PAE12 Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm target protein identification among precipitated proteins

• Cross-Species Reactivity Analysis:

  • Test on samples from species with known sequence homology

  • Reactivity should correlate with sequence conservation

  • PAE112Hu01 exhibits reactivity with human, mouse, and pig EFNB2

Validation Documentation:
Document all validation steps with appropriate controls in a systematic validation report. Include positive and negative controls for each experiment, details of experimental conditions, and quantitative analysis where possible.

This multi-method validation approach provides robust evidence of antibody specificity, increasing confidence in experimental results.

What sample preparation techniques maximize the detection sensitivity of PAE12 Antibody?

Optimized sample preparation is crucial for detecting low-abundance targets with maximum sensitivity:

Western Blotting Sample Preparation:

• Efficient Protein Extraction:

  • Use appropriate lysis buffers containing protease inhibitors

  • Optimize physical disruption methods (sonication, homogenization)

  • Maintain cold temperatures throughout processing

  • For membrane proteins, include appropriate detergents (Triton X-100, CHAPS, NP-40)

• Sample Processing:

  • Accurate protein quantification (BCA or Bradford assay)

  • Equal loading (15-30 μg total protein per lane typically)

  • Complete denaturation with SDS and heat (95°C for 5 minutes)

  • Include reducing agents (DTT or β-mercaptoethanol) if target contains disulfide bonds

  • Use freshly prepared samples when possible

Immunohistochemistry/Immunocytochemistry Optimization:

• Fixation Protocol:

  • Test multiple fixatives (4% paraformaldehyde, methanol/acetone)

  • Optimize fixation time (typically 10-30 minutes)

  • For some epitopes, light fixation may preserve antigenicity better

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Test multiple retrieval times and temperatures

  • For some antibodies, enzymatic retrieval (proteinase K, trypsin) may be superior

  • Cool slides slowly to room temperature after retrieval

• Blocking and Permeabilization:

  • Block with 5% normal serum from secondary antibody species

  • Include 0.1-0.3% Triton X-100 for intracellular antigens

  • Extended blocking (1-2 hours at room temperature or overnight at 4°C)

Immunoprecipitation Optimization:

• Pre-clearing Strategies:

  • Pre-clear lysates with protein A/G beads

  • Pre-incubate with blocking agents (e.g., anti-CD32 to block Fc receptors)

  • Use species-matched normal IgG for pre-clearing

• Antibody Coupling:

  • Cross-link antibody to beads to prevent co-elution

  • Use appropriate coupling chemistry (e.g., CNBr-activated Sepharose)

  • Optimize antibody-to-bead ratio

• Washing Optimization:

  • Balance between stringency and retention of specific interactions

  • Typically 3-5 washes with decreasing detergent concentrations

  • Include salt (150-500 mM NaCl) to reduce non-specific ionic interactions

These methodological refinements can significantly enhance detection sensitivity across different applications with PAE12 Antibody.

How can phage display technology improve PAE12 Antibody specificity?

Phage display technology offers powerful approaches for antibody specificity enhancement:

Comprehensive Phage Display Protocol:

• Library Construction:

  • Create a phage display library expressing variant forms of PAE12 antibody fragments

  • Focus on scFv, Fab, or VHH domains which display efficiently on phage

  • Target complementarity-determining regions (CDRs) for mutagenesis

  • Design diversity to explore sequence space around key binding residues

• Multi-Stage Biopanning Strategy:

• Advanced Selection Techniques:

  • Perform selections against multiple related antigens to identify differential binders

  • Include pre-selections to deplete undesired binders

  • Implement subtractive panning against closely related proteins

• High-Throughput Screening:

  • Screen selected clones against both target and related proteins

  • Use ELISA, fluorometric microvolume assay technology (FMAT), or chromophore-assisted laser inactivation (CALI)

  • Select clones with high target:cross-reactivity ratios

• Computational Analysis:

  • Sequence selected clones and analyze enriched mutations

  • Identify binding modes associated with specific target recognition

  • Apply energy minimization to predict binding properties:

    • For specific binders: Minimize energy function (E) for desired ligand while maximizing for undesired ligands

    • For cross-reactive binders: Jointly minimize energy functions for all desired targets

• Validation and Characterization:

  • Express selected antibody variants as soluble proteins

  • Characterize binding properties using surface plasmon resonance

  • Validate specificity across different applications (WB, IHC, IP)

  • Confirm improved specificity profile compared to original PAE12

This advanced phage display approach can generate PAE12 variants with substantially improved specificity for research applications requiring discrimination between closely related targets.