SH2D3A Antibody, FITC conjugated

What is SH2D3A protein and why is it targeted in research?

SH2D3A (SH2 domain containing 3A), also known as novel SH2-containing protein 1 (NSP1), is a 576 amino acid protein that plays a role in JNK activation . The protein contains one SH2 domain that binds to tyrosine-phosphorylated regions of target proteins, frequently linking activated growth factors to putative signal transduction proteins . SH2D3A interacts with p130 Cas and is found at low levels in fetal kidney, fetal lung, placenta, adult pancreas, kidney, and lung . It is subject to post-translational phosphorylation on multiple tyrosine residues and the gene encoding SH2D3A maps to human chromosome 19 . SH2D3A has also been shown to interact with the epidermal growth factor receptor . The protein is of interest in studies of cellular signaling pathways and potentially in cancer research due to its involvement in growth factor signaling.

What applications are appropriate for FITC-conjugated SH2D3A antibodies?

FITC-conjugated SH2D3A antibodies are primarily used in applications requiring fluorescent detection:

The choice of application should consider the subcellular localization of SH2D3A, which varies by isoform. While some isoforms (α, β, γ) localize primarily in the cytoplasm and at the plasma membrane, the δ isoform has been found to localize primarily to nucleoli , which may affect detection strategy.

What are optimal storage conditions for maintaining FITC-conjugated antibody activity?

FITC-conjugated antibodies require special handling to preserve both antibody integrity and fluorophore activity:

• Store at -20°C for long-term preservation

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles, which can degrade both antibody and fluorophore

• Store in a buffer containing glycerol (typically 50%) to prevent freezing damage

• Protect from light at all times to prevent photobleaching of the FITC fluorophore

• Some preparations include sodium azide (0.02-0.05%) as a preservative, though this should be considered when using in live cell applications due to potential cytotoxicity

Most manufacturers recommend a shelf life of approximately one year when stored properly, though activity should be validated before critical experiments .

How can I determine the optimal antibody concentration for my specific experimental conditions?

Optimizing antibody concentration involves systematic titration:

• Perform a dilution series (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000) of the FITC-conjugated SH2D3A antibody

• Include both positive control samples (cells/tissues known to express SH2D3A) and negative controls

• For immunofluorescence applications, start with manufacturer's recommendations, typically in the range of 1-5 μg/mL

• For flow cytometry, begin with 10-20 μg/mL as a starting point

• Evaluate signal-to-background ratio at each concentration

• Select the dilution that provides maximum specific signal with minimal background

The optimal concentration may vary depending on:

• Expression level of SH2D3A in your specific sample

• Fixation and permeabilization methods

• Detection system sensitivity

• Specific epitope accessibility, which can be influenced by protein conformation and interactions

What controls should be included when using FITC-conjugated SH2D3A antibodies?

A robust experimental design should include multiple controls:

For quantitative applications, consider including calibration standards with known fluorophore numbers per particle to enable standardization between experiments.

How does epitope selection affect SH2D3A detection in different cellular compartments?

The choice of epitope targeted by the SH2D3A antibody significantly impacts detection results:

• N-terminal epitopes (AA 1-300): Better for detecting full-length protein in Western blots, but may miss truncated isoforms

• Internal region epitopes (AA 211-231): Used in many commercial antibodies and suitable for multiple applications

• C-terminal epitopes (near C terminus): May detect specific isoforms but can be blocked in protein complexes

Different cellular compartments may affect epitope accessibility:

• The unique C-terminal tail of SH2D3A δ isoform contains nuclear localization sequences (NLS2 and NLS3) , which can impact antibody accessibility when the protein is in the nucleus

• Phosphorylation state of tyrosine residues may mask certain epitopes, particularly in signaling-active cells

• SH2 domain interactions with phosphorylated target proteins may conceal epitopes in this region

For comprehensive detection across cellular compartments, antibodies targeting conserved, accessible epitopes or using multiple antibodies targeting different regions are recommended.

What are the optimal fixation and permeabilization methods for SH2D3A immunofluorescence?

The choice of fixation and permeabilization significantly impacts SH2D3A detection:

For permeabilization:

• Triton X-100 (0.1-0.5%): Effective for nuclear proteins and suitable for detecting nucleolar SH2D3A δ isoform

• Saponin (0.1%): Gentler permeabilization, better for membrane-associated SH2D3A forms

• Digitonin (50 μg/mL): Selective permeabilization of plasma membrane, preserving nuclear envelope

One effective protocol for SH2D3A detection combines:

• Fixation with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization with 0.1% Triton X-100 for 10 minutes

• Blocking with 1-5% BSA for 30-60 minutes prior to antibody incubation

How can F/P (fluorescein to protein) ratio be optimized for maximum sensitivity in SH2D3A detection?

The F/P ratio is critical for optimal fluorescence detection and can be adjusted for specific applications:

• Typical commercial FITC-conjugated antibodies have F/P ratios of 3:1 to 8:1

• Higher F/P ratios increase sensitivity but may cause self-quenching and increase non-specific binding

• Lower F/P ratios produce cleaner signals but may lack sensitivity for low-abundance proteins

For custom conjugation of SH2D3A antibodies:

• Start with a purified antibody (>95% purity, typically protein G purified)

• Use a standardized conjugation protocol such as the FluoroTag FITC Conjugation Kit

• Measure the F/P ratio spectrophotometrically after conjugation:

  • A₄₉₅/A₂₈₀ ratio indicates labeling efficiency

  • Optimal F/P ratio depends on the specific application; 4:1 is often a good compromise

• For low-abundance SH2D3A detection, higher F/P ratios (6-8:1) may be necessary

• For multiplexing with other fluorophores, lower F/P ratios (2-3:1) may reduce bleed-through

Commercial FITC-conjugated SH2D3A antibodies typically specify their F/P ratio and optimization has already been performed.

How do different SH2D3A isoforms and their post-translational modifications affect antibody binding?

SH2D3A has multiple isoforms with different subcellular localizations:

Post-translational modifications affecting antibody binding:

• Tyrosine phosphorylation: SH2D3A is subject to phosphorylation on multiple tyrosine residues , which may mask epitopes

• Protein-protein interactions: The SH2 domain binding to phospho-tyrosine targets may shield epitopes in this region

• Conformational changes: JNK activation may induce conformational changes affecting epitope accessibility

For comprehensive isoform detection:

• Use antibodies targeting conserved regions present in all isoforms

• When studying specific isoforms, select antibodies targeting isoform-unique regions

• For phosphorylation studies, use either phospho-specific antibodies or general antibodies under denaturing conditions

• Consider the R680C variant in the unique C-terminal tail of SH2D3A δ, which affects dendritic complexity and may alter antibody binding

What strategies can minimize cross-reactivity with other SH2 domain-containing proteins?

Cross-reactivity is a significant concern with SH2 domain-containing proteins due to conserved structural elements:

• Epitope selection is critical:

  • Choose antibodies generated against unique regions outside the conserved SH2 domain

  • Several commercial antibodies target internal regions (AA 211-231) or C-terminal regions with lower homology

  • Avoid antibodies targeting the highly conserved SH2 domain unless specificity has been rigorously validated

• Validation strategies to confirm specificity:

  • Western blot analysis should show a single band at approximately 63 kDa (the predicted size of SH2D3A)

  • Peptide competition assays using the immunizing peptide (e.g., APRAERFEKFQR)

  • Testing on samples with SH2D3A knockdown or knockout

  • Testing for cross-reactivity against recombinant SH2 domains from related proteins

• Additional controls:

  • Include samples expressing known levels of SH2D3A

  • Use the PolyMap (polyclonal mapping) high-throughput method for mapping protein-protein interactions to identify potential cross-reactivities

  • Compare results with orthogonal detection methods (e.g., RNA expression data from The Human Protein Atlas)

What are the considerations for multiplexing FITC-conjugated SH2D3A antibodies with other fluorophores?

Successful multiplexing requires careful planning to avoid spectral overlap and maximize signal separation:

• Spectral considerations:

  • FITC has excitation maximum at 495 nm and emission maximum at 525 nm

  • Choose compatible fluorophores with minimal spectral overlap, such as:

    • DAPI (blue): Ex 358 nm / Em 461 nm for nuclear counterstaining

    • TRITC (red): Ex 557 nm / Em 576 nm for a second protein of interest

    • Cy5 (far-red): Ex 650 nm / Em 670 nm for a third protein of interest

• Antibody selection for co-staining:

  • Use antibodies from different host species to avoid cross-reactivity (e.g., rabbit anti-SH2D3A with mouse anti-target 2)

  • If using same-species antibodies, consider directly conjugated formats or sequential staining protocols

• Controls for multiplexed experiments:

  • Single-color controls to establish compensation settings

  • Fluorescence minus one (FMO) controls to set accurate gating boundaries

  • Isotype controls for each fluorophore and species

• Instrument considerations:

  • For flow cytometry: Ensure appropriate laser and filter sets for each fluorophore

  • For microscopy: Use narrow bandpass filters to minimize bleed-through

  • Consider spectral unmixing for confocal applications with significant overlap

• Experimental protocol adjustments:

  • Increase washing steps between antibody incubations to reduce background

  • Optimize fixation protocols to preserve all target epitopes

  • Consider sequential rather than simultaneous staining for challenging targets

How is FITC-conjugated SH2D3A antibody used in current research on neuronal signaling pathways?

Recent research has revealed important roles for SH2D3A in neuronal development and signaling:

• Neuronal architecture studies:

  • SH2D3A isoforms (particularly δ) have been shown to impact neuronal complexity and dendritic branching

  • FITC-conjugated antibodies enable visualization of differential subcellular localization of isoforms in primary neurons

  • Live imaging using directly conjugated antibodies allows temporal tracking of SH2D3A redistribution during neuronal development

• Methodological approaches:

  • Primary neuronal cultures transfected with GFP-tagged SH2B1 isoforms have revealed that specific isoforms enhance NGF-induced neurite outgrowth

  • FITC-conjugated SH2D3A antibodies provide a complementary approach to visualize endogenous protein distribution

  • Studies comparing GFP-tagged and antibody-detected distributions help validate localization patterns and avoid overexpression artifacts

• Variant analysis in neuronal function:

  • Obesity-associated SH2B1 variants including A484T and R680C affect neurite complexity and have been studied using fluorescent antibody approaches

  • FITC-conjugated antibodies against SH2D3A enable investigation of similar structure-function relationships

• Experimental design considerations:

  • For neuronal studies, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is typically effective

  • Image analysis software can quantify colocalization of SH2D3A with synaptic markers or other signaling components

  • Time-course experiments can track changes in SH2D3A localization during neuronal stimulation or development

What are the latest approaches for validating FITC-conjugated SH2D3A antibody specificity?

The emergence of new technologies has enhanced antibody validation strategies:

• High-throughput specificity profiling:

  • The PolyMap method allows mapping of protein-protein interactions using a surface display platform with robust expression of single antigens per cell

  • This approach can be applied to validate SH2D3A antibodies by testing against libraries of SH2 domain-containing proteins

  • The method combines unique barcoding for antigen identification with simplified cloning processes

• Gene editing-based validation:

  • CRISPR/Cas9-mediated knockout of SH2D3A provides definitive negative controls

  • Comparing staining patterns between wild-type and knockout samples confirms specificity

  • Genetic tagging of endogenous SH2D3A (e.g., with HaloTag) provides orthogonal validation of antibody-detected localization

• Mass spectrometry validation:

  • Immunoprecipitation with the SH2D3A antibody followed by mass spectrometry confirms target identity

  • Comparing detected peptides with the antibody's target epitope verifies specific recognition

  • Quantitative proteomics can assess off-target binding

• Advanced imaging approaches:

  • Super-resolution microscopy techniques (STORM, PALM, STED) provide nanoscale validation of antibody specificity by revealing expected subcellular distributions

  • Proximity ligation assays confirm interactions with known binding partners, such as p130 Cas or BCAR1

  • F-techniques (FRET, FLIM, FRAP) can validate functional aspects of the detected protein

How can researchers ensure reproducibility when using different lots of FITC-conjugated SH2D3A antibodies?

Antibody lot-to-lot variation is a significant challenge in research reproducibility:

• Standardization practices:

  • Maintain detailed records of antibody source, catalog number, lot number, and validation data

  • Develop internal standard operating procedures (SOPs) for antibody validation

  • Consider creating a reference sample set that all new antibody lots must be tested against

• Quantitative validation methods:

  • Determine F/P ratio for each lot using spectrophotometric analysis:

    • Measure absorbance at 280 nm (protein) and 495 nm (FITC)

    • Calculate molar F/P ratio using the formula: F/P = (A495 × dilution factor)/(195 × [protein concentration in mg/mL])

  • Compare staining intensity using standardized samples and image acquisition settings

  • Use calibration beads with known fluorescence intensities to standardize flow cytometry data

• Documentation and reporting:

  • Include comprehensive antibody information in publications following the Minimum Information About Antibody Validation (MIAV) guidelines

  • Share validation data through antibody validation repositories

  • Document optimization steps for new lots in laboratory notebooks

• Alternative strategies when facing lot variation:

  • Purchase multiple vials of well-performing lots for critical long-term projects

  • Consider conjugating unconjugated antibodies in-house using standardized FITC conjugation kits

  • Use orthogonal detection methods to validate key findings