SH2D3A Antibody, HRP conjugated

What is SH2D3A and why is it a subject of research interest?

SH2D3A (SH2 Domain Containing 3A) is a human protein also known as NSP1 (Novel SH2-containing protein 1) . This 63 kDa protein contains an SH2 domain that typically binds to phosphorylated tyrosine residues on other proteins, suggesting its involvement in signal transduction pathways. Research indicates it may play a significant role in JNK activation , making it relevant to studies of cellular stress responses, inflammation, and potentially oncogenic signaling.

The protein's structure includes specific regions that serve as epitopes for antibody generation, with commercially available antibodies targeting various segments including AA 1-576 (full length), AA 460-575, AA 211-231, and other internal regions . Researchers study SH2D3A to elucidate its function in signaling networks and potential implications in disease processes.

Horseradish peroxidase (HRP) conjugation involves chemically linking this 44 kDa glycoprotein to antibodies through a process that targets the 6 lysine residues on HRP . The classical conjugation method follows these principles:

• Activation of HRP carbohydrate moieties using sodium meta periodate to generate aldehyde groups through oxidation .

• Reaction of these aldehyde groups with amino groups on the antibody to form Schiff's bases.

• Stabilization of these bonds through reduction using sodium cyanoborohydride .

The modified protocols incorporate an additional lyophilization (freeze-drying) step after HRP activation, which:

• Reduces reaction volume without altering reactant quantities

• Increases collision frequency between activated HRP and antibody molecules

• Enhances conjugation efficiency by promoting better molecular interactions

This process creates a stable covalent linkage between the enzyme and antibody while preserving both the enzymatic activity of HRP and the antigen-binding capability of the antibody .

What are the recommended applications for HRP-conjugated SH2D3A antibodies?

HRP-conjugated SH2D3A antibodies are versatile tools for various immunological techniques:

Western Blotting (WB)

• Recommended dilutions: 1:100-1000

• Detects SH2D3A at approximately 63 kDa

• Validated in human cell lines including RT-4 and U-251 MG

Enzyme-Linked Immunosorbent Assay (ELISA)

• High sensitivity with dilutions ranging from 1:100-1000

• Direct detection without secondary antibody requirement

• Reduced background compared to two-step detection systems

Immunohistochemistry (IHC)

• Recommended dilutions: 1:100-500

• Works with paraffin-embedded tissue sections

• Produces a brown precipitate with DAB substrate

Immunofluorescence (IF)

• Validated at 4 μg/mL concentration

• Effective in paraformaldehyde-fixed, Triton X-100 permeabilized cells

• Can be visualized when used with appropriate fluorogenic HRP substrates

The direct conjugation approach eliminates cross-species reactivity concerns and reduces protocol time by removing secondary antibody incubation steps .

How can researchers validate the successful conjugation of HRP to SH2D3A antibodies?

Multiple analytical methods should be employed to confirm successful HRP conjugation to SH2D3A antibodies:

UV-Visible Spectroscopy

Perform wavelength scanning (280-800 nm) and compare:

• Unconjugated antibody shows absorption peak at 280 nm

• Unconjugated HRP shows characteristic peak at 430 nm

• Successfully conjugated product displays a modified absorption profile with a shift at 430 nm region, confirming chemical modification

SDS-PAGE Analysis

• Heat-denatured conjugates (95°C) show altered migration patterns

• Unconjugated components (antibody and HRP separately) show distinct bands

• Successfully conjugated products often show limited mobility due to increased molecular weight

Functional Validation

• Direct ELISA using known SH2D3A-containing samples

• Assessment of signal-to-noise ratio

• Comparison with unconjugated primary antibody + HRP-labeled secondary antibody system

• Dilution series to determine working concentration range

These validation steps ensure both retained enzymatic activity and preserved antigen recognition capability of the conjugated product.

What factors influence the sensitivity of HRP-conjugated SH2D3A antibodies in immunoassays?

Multiple factors determine the sensitivity of HRP-conjugated SH2D3A antibodies:

Conjugation Method Impact

Research demonstrates that a modified periodate method incorporating lyophilization significantly enhances sensitivity:

• Conjugates prepared with modified methods show effectiveness at dilutions up to 1:5000

• Classical conjugation methods typically require much lower dilutions (around 1:25)

• Statistical analysis shows highly significant difference (p < 0.001) between methods

Substrate Selection

Different substrates provide varying levels of sensitivity:

• DAB (diaminobenzidine): Produces brown precipitate, moderate sensitivity

• TMB (3,3',5,5'-tetramethylbenzidine): Higher sensitivity, produces blue color

• Enhanced chemiluminescent substrates: Highest sensitivity for applications like Western blotting

Assay Conditions

• Incubation time and temperature optimization

• Buffer composition effects on enzymatic activity

• Blocking reagent selection to minimize background

• Sample preparation techniques to maximize target availability

Conjugate Stabilization

• Use of preservatives like LifeXtend™ HRP conjugate stabilizer

• Protection from factors that can reduce conjugate performance

• Proper storage conditions to maintain activity

The enhanced sensitivity of optimized conjugates enables detection of lower amounts of SH2D3A, which can be crucial for detecting subtle biological changes in research contexts.

What are the best practices for storage and handling of HRP-conjugated SH2D3A antibodies?

To maintain optimal activity of HRP-conjugated SH2D3A antibodies, researchers should follow these guidelines:

Storage Conditions

• Store at 4°C in dark conditions

• Expected shelf-life is typically up to 6 months under proper storage

• Avoid repeated freeze-thaw cycles that can compromise both antibody and enzyme activity

• Store in appropriate buffer formulation (e.g., 0.01M Sodium Phosphate, 0.25M NaCl, pH 7.6)

Stabilizing Additives

• BSA (5mg/ml) helps prevent non-specific adsorption and stabilizes the protein

• Sodium azide (0.02%) prevents microbial contamination

• Consider specialized stabilizers for extended shelf-life

Working Solution Preparation

• Prepare fresh dilutions for each experiment whenever possible

• Use sterile buffers free of contaminants

• Avoid exposure to strong oxidizing agents that may inactivate HRP

• Minimize exposure to light and heat during handling

Quality Control Monitoring

• Periodically test aliquots for activity retention

• Include positive controls in experiments to verify conjugate performance

• Document lot-to-lot variations when using commercial preparations

Proper storage and handling significantly impact experimental reproducibility and reliability when working with these sensitive reagents.

How can researchers troubleshoot weak or non-specific signals when using HRP-conjugated SH2D3A antibodies?

When encountering signal issues with HRP-conjugated SH2D3A antibodies, systematic troubleshooting should address:

Weak Signal Resolution

• Antibody concentration: Titrate to optimal working dilution (typical range 1:100-1:1000 for WB)

• Sample preparation: Ensure adequate protein extraction and denaturation

• Incubation conditions: Extend time or adjust temperature

• Detection system: Try higher sensitivity substrates or enhanced chemiluminescence

• Antibody storage: Check for potential activity loss due to improper storage

• Antigen retrieval: Optimize methods for fixed samples to improve epitope accessibility

Non-specific Signal Troubleshooting

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers)

• Washing stringency: Increase wash duration or detergent concentration

• Antibody specificity: Validate with positive and negative controls

• Endogenous peroxidase activity: Include quenching steps (e.g., hydrogen peroxide treatment)

• Buffer composition: Adjust salt concentration to increase specificity

• Cross-reactivity: Pre-absorb antibody against potential cross-reactive proteins

Controls Implementation

• Positive control: Samples known to express SH2D3A (e.g., RT-4 or U-251 MG cell lines)

• Negative control: Samples without SH2D3A expression

• Isotype control: Non-specific antibody of the same isotype and conjugate

• Substrate control: Sample with substrate only to check for endogenous peroxidase

Systematic evaluation of these parameters should identify the source of signal problems and guide appropriate adjustments.

What are the advantages of HRP-conjugated antibodies compared to other detection systems for SH2D3A research?

HRP-conjugated SH2D3A antibodies offer several advantages over alternative detection approaches:

Comparison with Two-Step Detection Systems

• Elimination of secondary antibody incubation steps saves time

• Reduced background from cross-reactivity of secondary antibodies

• Fewer washing steps resulting in shorter protocols

• Direct detection provides more consistent results between experiments

Sensitivity Enhancement

• Modified conjugation protocols demonstrate significantly improved sensitivity:

  • Working dilutions of 1:5000 vs 1:25 with classical methods

  • Enhanced ability to detect lower amounts of target protein

Signal Amplification Options

• Compatibility with tyramide signal amplification (TSA) for further sensitivity enhancement

• Various substrate options allowing customization based on application needs

• Enzymatic reaction provides natural signal amplification through multiple substrate conversions per enzyme molecule

Practical Research Considerations

• Cost-effectiveness for high-throughput applications

• Reduced potential for cross-species reactivity in complex biological samples

• Compatibility with multiplex staining when using appropriate substrates

• Enhanced ability for early diagnosis of diseases through detection of low abundance biomarkers

These advantages make HRP-conjugated antibodies particularly valuable for detailed characterization of SH2D3A expression and interactions in various biological contexts.

How do different fixation and permeabilization methods affect the performance of HRP-conjugated SH2D3A antibodies?

Fixation and permeabilization protocols significantly impact the performance of HRP-conjugated SH2D3A antibodies:

Fixation Method Effects

• Paraformaldehyde fixation (4%) has been validated with SH2D3A antibodies for immunofluorescence applications

• Different epitopes may show variable sensitivity to fixation methods:

  • N-terminal epitopes often more sensitive to fixation-induced masking

  • Internal regions may require more stringent permeabilization

  • C-terminal epitopes (like those targeted by ABIN570964) may have different accessibility profiles

Permeabilization Considerations

• Triton X-100 permeabilization has been demonstrated effective for SH2D3A detection

• Optimization parameters include:

  • Concentration (typically 0.1-0.5%)

  • Incubation time (5-30 minutes)

  • Temperature (room temperature vs. 4°C)

Antigen Retrieval Importance

• Heat-induced epitope retrieval may be necessary for formalin-fixed samples

• Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) methods should be compared

• Enzymatic retrieval (e.g., proteinase K) may be required for heavily fixed samples

Protocol Optimization Strategy

• Compare multiple fixation methods (paraformaldehyde, methanol, acetone)

• Test different permeabilization reagents (Triton X-100, Tween-20, saponin)

• Evaluate antigen retrieval methods if using archived fixed samples

• Validate with positive control samples expressing SH2D3A

Researchers should systematically optimize these parameters for each specific SH2D3A antibody clone and application to achieve optimal signal-to-noise ratio.

What methodological considerations are important when designing experiments with HRP-conjugated SH2D3A antibodies?

Experimental design with HRP-conjugated SH2D3A antibodies requires careful attention to several methodological aspects:

Antibody Selection Based on Target Epitope

• Choose antibodies targeting specific regions based on research question:

  • Full-length antibodies (AA 1-576) for general detection

  • Domain-specific antibodies for functional studies

  • Phospho-specific antibodies if studying activation state

  • Consider epitope conservation if studying non-human models

Control Implementation

• Positive controls: Cell lines with confirmed SH2D3A expression (RT-4, U-251 MG)

• Negative controls: Cell lines with low/no expression or knockdown models

• Blocking peptide controls: Pre-incubation with immunizing peptide

• Loading controls for quantitative comparisons

Quantification Approaches

• Densitometry for Western blot quantification

• Mean fluorescence intensity measurements for immunofluorescence

• Establish standard curves using recombinant SH2D3A protein for absolute quantification

• Normalization strategies to account for experimental variations

Multiplex Considerations

• If combining with other antibodies, consider:

  • Species compatibility to avoid cross-reactivity

  • Distinct visualization methods for each target

  • Sequential detection protocols if using multiple HRP conjugates

  • Careful validation of specificity in multiplex context

Application-Specific Optimization

• Western blotting: Determine optimal protein loading (typically 20-50 μg total protein)

• ELISA: Establish coating conditions and blocking parameters

• Immunohistochemistry: Optimize section thickness and antigen retrieval

• Immunofluorescence: Adjust exposure settings to prevent photobleaching of fluorogenic HRP substrates

Thoughtful consideration of these methodological aspects ensures generation of reliable, reproducible data when working with HRP-conjugated SH2D3A antibodies.

How can researchers validate the specificity of HRP-conjugated SH2D3A antibodies?

Validating antibody specificity is crucial for generating reliable research data. For HRP-conjugated SH2D3A antibodies, researchers should implement multiple validation approaches:

Genetic Validation Approaches

• siRNA/shRNA knockdown: Compare signal in knockdown vs. control cells

• CRISPR/Cas9 knockout: Eliminate target gene expression for absolute negative control

• Overexpression studies: Analyze signal in cells with forced SH2D3A expression

• These genetic approaches provide the most definitive specificity validation

Biochemical Validation Methods

• Western blot molecular weight confirmation: SH2D3A should appear at ~63 kDa

• Peptide competition: Pre-incubation with immunizing peptide should abolish signal

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-validation with multiple antibodies targeting different epitopes

Cellular Validation Techniques

• Immunofluorescence pattern analysis: Subcellular localization should be consistent with known biology

• Co-localization with established markers of expected compartments

• Signal correlation with known expression patterns across cell types

• Comparison with validated antibodies from different suppliers

Application-Specific Validation

• For Western blotting: Check for single band at expected molecular weight

• For ELISA: Demonstrate dose-dependent signal with recombinant protein

• For IHC/IF: Compare staining patterns with published literature and RNA expression data

• For all applications: Include appropriate positive and negative controls

These comprehensive validation strategies ensure that experimental findings reflect true SH2D3A biology rather than artifacts from non-specific antibody reactivity.