sh3pxd2a Antibody

What is SH3PXD2A and why is it important in research?

SH3PXD2A, also known as Tks5, is an adapter protein involved in invadopodia and podosome formation, extracellular matrix degradation, and invasiveness of cancer cells. It binds matrix metalloproteinases (ADAMs), NADPH oxidases (NOXs), and phosphoinositides . As a key player in cell signaling pathways that regulate cytoskeletal dynamics and cell movement, SH3PXD2A is crucial for studying cancer metastasis, invasiveness, and cellular behaviors involving migration . The protein contains an amino-terminal PX domain followed by five SH3 domains and is typically cytoplasmic in normal fibroblasts .

Genetic studies have revealed that homozygous disruption of the sh3pxd2a gene in mice resulted in neonatal death and the presence of complete cleft of the secondary palate in some cases, highlighting its developmental importance .

Most commercial SH3PXD2A antibodies should be stored at -20°C for long-term stability . The common storage buffer is PBS with 0.02% sodium azide and 50% glycerol pH 7.3 . According to manufacturer recommendations, these antibodies are typically stable for one year after shipment when stored properly .

For antibodies stored at -20°C, aliquoting is generally unnecessary, although this may vary between manufacturers . Upon receipt, antibodies are typically shipped on wet ice and should be transferred to the recommended storage conditions immediately .

How do I validate the specificity of a new SH3PXD2A antibody?

Validating specificity is crucial before conducting critical experiments:

• Positive Control Selection: Use cell lines known to express SH3PXD2A such as HeLa or HepG2 cells .

• Knockout/Knockdown Validation: Test the antibody in SH3PXD2A knockout or knockdown samples. Several publications have used this method for validation .

• Western Blot Analysis: Verify that the antibody detects bands at the expected molecular weight. SH3PXD2A has a calculated molecular weight of 125 kDa but is typically observed at 140-150 kDa in Western blots .

• Cross-reactivity Testing: If working with non-human samples, ensure the antibody has confirmed reactivity with your species of interest. Many SH3PXD2A antibodies show reactivity with human, mouse, and rat samples .

• Immunogen Sequence Analysis: Check if the immunogen sequence used to generate the antibody has homology with your target species. For example, PA5-58168 antibody has 90% sequence identity with mouse and 89% with rat orthologs .

What controls should be included when studying SH3PXD2A in cancer models?

When investigating SH3PXD2A in cancer research, incorporate these essential controls:

• Normal Tissue/Cell Controls: Include normal counterparts to cancer tissues/cells to establish baseline expression levels.

• Genetic Controls: Use SH3PXD2A knockout or knockdown models. Homozygous disruption of the sh3pxd2a gene has shown phenotypic manifestations including neonatal death and cleft palate .

• Secondary Antibody Controls: Include samples processed without primary antibody to assess non-specific binding.

• Isotype Controls: Use rabbit IgG (matching the SH3PXD2A antibody host species) at equivalent concentrations to rule out non-specific binding.

• Functional Controls: When studying invasion or migration, include known inhibitors of these processes as positive controls.

How can I detect different isoforms of SH3PXD2A?

SH3PXD2A exists in multiple forms, including p140 and p130, which may be generated by alternative splicing . To study these isoforms:

• Select Appropriate Antibodies: Choose antibodies targeting regions common to all isoforms or isoform-specific regions. For instance, antibodies targeting the N-terminal region (like OAAB11978) may detect different isoforms than those targeting C-terminal regions .

• 5′ RACE Analysis: To identify new transcription initiation sites, perform 5′ RACE as described in the literature. This technique has been used to identify alternative transcription start sites in the sh3pxd2a gene .

• Western Blot Optimization:

  • Use gradient gels (4-12%) for better separation of high molecular weight proteins

  • Extend transfer time for large proteins

  • Consider using specialized buffers for optimal resolution of isoforms

• PCR-based Approaches: Design primers targeting specific exons to detect alternative splicing variants at the mRNA level before protein analysis.

What are the best approaches for studying SH3PXD2A's role in invadopodia formation and cancer cell invasion?

To investigate SH3PXD2A's functions in cancer invasion mechanisms:

• Gelatin Degradation Assays: Plate cells on fluorescently-labeled gelatin matrix to visualize and quantify matrix degradation associated with invadopodia formation.

• Co-localization Studies: Use immunofluorescence to examine co-localization of SH3PXD2A with other invadopodia markers (cortactin, Arp2/3, etc.) using the recommended IF dilutions (1:10-1:100) .

• 3D Invasion Assays: Employ Matrigel invasion chambers to assess the effect of SH3PXD2A manipulation on invasion capacity.

• Protein Interaction Studies: Use co-immunoprecipitation to study SH3PXD2A interactions with matrix metalloproteinases and NADPH oxidases. SH3PXD2A antibodies have been validated for CoIP applications .

• ROS Detection Assays: Since SH3PXD2A acts as an organizer protein allowing NOX1- or NOX3-dependent reactive oxygen species generation, include ROS detection methods (such as DCF-DA fluorescence) in your experimental design .

How do I investigate the interaction between SH3PXD2A and ADAM12 in mediating amyloid-beta peptide neurotoxicity?

To study this specific interaction in neurotoxicity contexts:

• Co-immunoprecipitation: Use anti-SH3PXD2A antibodies to pull down protein complexes, then probe for ADAM12, or vice versa. The antibody dilution should be optimized for CoIP applications .

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity.

• Functional Assays: Design experiments with the following components:

  • Neuronal cell cultures treated with amyloid-beta peptide

  • SH3PXD2A and/or ADAM12 knockdown/knockout

  • Cell viability and apoptosis measurements

  • Rescue experiments with wild-type or mutant constructs

• Domain Mapping: Create truncation or point mutation constructs of SH3PXD2A to identify which domains (PX or specific SH3 domains) are essential for ADAM12 interaction and amyloid-beta-mediated neurotoxicity.

Why might Western blots with SH3PXD2A antibodies show unexpected band patterns?

Several factors can contribute to unexpected Western blot results:

• Multiple Isoforms: SH3PXD2A has multiple forms (p140 and p130) that may appear as distinct bands .

• Post-translational Modifications: Phosphorylation or other modifications can cause shifts in apparent molecular weight.

• Sample Preparation: Inadequate lysis or protein degradation can result in additional bands. Use protease inhibitors in lysis buffers.

• Antibody Specificity: Some antibodies may cross-react with related proteins. Verify specificity using knockout/knockdown controls.

• Technical Considerations:

  • For optimal results, follow recommended dilutions (typically 1:500-1:1000 for Western blot)

  • Use freshly prepared samples

  • Consider gradient gels for better resolution of high-molecular-weight proteins

How can I optimize immunohistochemistry protocols for SH3PXD2A detection in tissue samples?

For optimal IHC results with SH3PXD2A antibodies:

• Antigen Retrieval: Use TE buffer pH 9.0 for optimal results, or alternatively, citrate buffer pH 6.0 .

• Antibody Dilution: Start with the recommended range (1:20-1:200) and optimize for your specific tissue type .

• Incubation Conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

• Signal Amplification: Consider using polymer-based detection systems for enhanced sensitivity.

• Positive Controls: Include tissues known to express SH3PXD2A, such as human breast cancer tissue .

• Background Reduction:

  • Block with appropriate serum (typically 5-10% from the same species as the secondary antibody)

  • Include 0.1-0.3% Triton X-100 in blocking solutions for improved penetration

  • Use hydrogen peroxide to block endogenous peroxidase activity

What are the current hypotheses regarding SH3PXD2A's role in ROS generation and cancer progression?

Current research suggests several mechanisms through which SH3PXD2A influences ROS generation and cancer progression:

• NOX Complex Assembly: SH3PXD2A acts as an organizer protein that facilitates NOX1- or NOX3-dependent reactive oxygen species generation and localization .

• Invadopodia Formation: SH3PXD2A is essential for the formation of invadopodia, specialized cellular protrusions that facilitate cancer cell invasion through the extracellular matrix .

• Signaling Integration: SH3PXD2A may serve as a scaffold that integrates multiple signaling pathways involved in cell migration, invasion, and ROS production.

• Microenvironment Modification: Through its role in ROS generation and matrix degradation, SH3PXD2A might contribute to remodeling the tumor microenvironment in ways that promote cancer progression.

• Therapeutic Target Potential: Emerging evidence suggests that targeting SH3PXD2A or its interactions could potentially inhibit cancer cell invasion and metastasis.

How can genetic approaches be used to study SH3PXD2A function in developmental and disease contexts?

Genetic approaches offer powerful tools for investigating SH3PXD2A:

• Mouse Models: Homozygous disruption of the sh3pxd2a gene in mice has revealed its importance in palate development, as these mice exhibit neonatal death and cleft palate .

• Conditional Knockout Systems: Tissue-specific and inducible knockouts can help dissect SH3PXD2A's function in specific contexts while avoiding developmental lethality.

• CRISPR/Cas9 Genome Editing: This technology allows precise modification of the SH3PXD2A gene to:

  • Create knockout cell lines for in vitro studies

  • Introduce specific mutations to study structure-function relationships

  • Generate reporter systems by tagging endogenous SH3PXD2A

• Domain-specific Mutations: Creating point mutations or deletions in specific domains (PX domain or individual SH3 domains) can help map the functional importance of each domain.

• Rescue Experiments: Reintroducing wild-type or mutant SH3PXD2A into knockout backgrounds can confirm specificity and reveal which domains are essential for specific functions.

The variable phenotypes observed in sh3pxd2a mutant mice (some dying within 24 hours with cleft palate, some dying later without cleft palate, and some reaching adulthood with no apparent phenotype) suggest complex genetic interactions that warrant further investigation.