SH3PXD2A Antibody

What is SH3PXD2A and what cellular functions does it regulate?

SH3PXD2A, also known as TKS5 (Tyrosine kinase substrate with five SH3 domains), is an adapter protein primarily involved in invadopodia and podosome formation, extracellular matrix degradation, and cancer cell invasion . This protein contains an amino-terminal PX domain followed by five SH3 domains and functions as a scaffold protein in both normal and transformed cell lines . SH3PXD2A binds matrix metalloproteinases (ADAMs), NADPH oxidases (NOXs), and phosphoinositides, acting as an organizer protein that facilitates NOX1- or NOX3-dependent reactive oxygen species (ROS) generation and localization . Additionally, in association with ADAM12, it mediates the neurotoxic effect of amyloid-beta peptide .

What is the molecular structure and weight of SH3PXD2A?

SH3PXD2A has a calculated molecular weight of approximately 125.289 kDa , though in Western blot applications it is typically observed at 140-150 kDa . This discrepancy between calculated and observed molecular weights may be attributed to post-translational modifications. The protein contains distinct functional domains: an N-terminal PX (phox homology) domain that binds phosphoinositides and five SH3 domains that mediate protein-protein interactions, particularly with proteins containing proline-rich regions .

How should I optimize Western blot protocols for detecting SH3PXD2A?

For Western blot detection of SH3PXD2A, consider the following optimization steps:

• Sample preparation: Use protein extraction methods that preserve phosphorylation states if studying post-translational modifications

• Gel percentage: Use 8-10% SDS-PAGE gels to achieve optimal separation of the 140-150 kDa protein

• Transfer conditions: Implement wet transfer at lower voltage (30V) overnight for better transfer of high molecular weight proteins

• Antibody dilution: Start with manufacturer-recommended dilutions (typically 1:500-1:1000) and optimize as needed

• Detection systems: Enhanced chemiluminescence (ECL) systems are generally suitable, but fluorescent secondary antibodies may provide better quantification

• Positive controls: HeLa and HepG2 cells have been confirmed to express detectable levels of SH3PXD2A

Researchers should note that the antibody may detect both p140 and p130 forms of SH3PXD2A, which may represent different splice variants .

What are the critical factors for successful immunohistochemistry with SH3PXD2A antibodies?

For optimal immunohistochemistry results with SH3PXD2A antibodies:

• Antigen retrieval: Use TE buffer pH 9.0 as suggested for many SH3PXD2A antibodies, though citrate buffer pH 6.0 may be used as an alternative

• Antibody dilution: Begin with 1:20-1:200 dilution and optimize based on signal-to-noise ratio

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations

• Detection systems: Both DAB and fluorescence-based detection systems have been validated

• Positive tissue controls: Human breast cancer tissue has been validated as a positive control for SH3PXD2A detection

• Blocking: Use appropriate blocking buffers containing BSA to minimize background staining

It is essential to include both positive and negative controls in each experiment to validate antibody specificity.

How do I troubleshoot non-specific binding or background issues with SH3PXD2A antibodies?

When encountering non-specific binding or high background:

• Increase blocking time/concentration: Use 3-5% BSA or 5-10% normal serum from the secondary antibody host species

• Increase washing steps: Add additional washes with PBS-T (0.1% Tween-20)

• Titrate primary antibody: Test a range of dilutions to find optimal signal-to-noise ratio

• Pre-absorb antibody: Incubate antibody with cell/tissue lysates from SH3PXD2A-negative samples

• Use affinity-purified antibodies: Select antibodies that have undergone affinity purification

• Reduce secondary antibody concentration: Dilute secondary antibody further if background persists

• Include appropriate controls: Use knockout or knockdown samples to confirm specificity

Remember that validation is sample-dependent, and optimization may be required for each experimental system .

How can I use SH3PXD2A antibodies to study invadopodia formation in cancer research?

To study invadopodia formation using SH3PXD2A antibodies:

• Co-localization studies: Perform immunofluorescence with SH3PXD2A antibodies alongside markers for F-actin (phalloidin) and cortactin to identify invadopodia structures

• Gelatin degradation assays: Culture cells on fluorescent gelatin, then use SH3PXD2A antibodies to correlate protein localization with matrix degradation sites

• Proximity ligation assays: Investigate SH3PXD2A interactions with key invadopodia components like matrix metalloproteinases

• Live-cell imaging: Use fluorescently tagged anti-SH3PXD2A antibody fragments to track invadopodia dynamics

• Knockdown validation: Employ siRNA or CRISPR-Cas9 SH3PXD2A knockdown/knockout as controls to validate antibody specificity

These approaches can help elucidate SH3PXD2A's role in cancer cell invasion and metastasis, particularly in breast cancer where SH3PXD2A expression has been linked to disease progression .

What considerations are important when studying SH3PXD2A's interaction with NADPH oxidases?

When investigating SH3PXD2A's role in NOX-dependent ROS generation:

• Co-immunoprecipitation: Use SH3PXD2A antibodies for pull-down experiments followed by blotting for NOX components

• ROS detection assays: Combine immunofluorescence staining of SH3PXD2A with fluorescent ROS indicators

• Domain-specific antibodies: Select antibodies targeting specific domains of SH3PXD2A to determine which regions interact with NOX proteins

• Subcellular fractionation: Isolate membrane fractions where active NOX complexes reside and probe for SH3PXD2A

• Phosphorylation state: Consider using phospho-specific antibodies as SH3PXD2A activity may be regulated by phosphorylation

• Control conditions: Include antioxidant treatments to verify ROS-dependent effects

These approaches can help determine how SH3PXD2A functions as an organizer protein in NOX1- or NOX3-dependent ROS generation and localization .

How can I differentiate between SH3PXD2A isoforms in my experimental system?

To distinguish between different SH3PXD2A isoforms or post-translational modifications:

• Epitope selection: Choose antibodies raised against epitopes that are unique to specific isoforms

• Immunogen comparison: Review the immunogen sequences for each antibody to understand which protein regions they target

• Gel resolution: Use gradient gels (4-15%) to improve separation of closely migrating isoforms

• Phosphatase treatment: Treat samples with phosphatases prior to Western blotting to identify modifications due to phosphorylation

• 2D gel electrophoresis: Separate proteins by both isoelectric point and molecular weight to distinguish isoforms

• Mass spectrometry validation: Confirm the identity of bands detected by the antibody using mass spectrometry

• Knockout controls: Use targeted knockout of specific isoforms to validate antibody specificity

The p140 and p130 forms of SH3PXD2A may represent different splice variants and require careful analysis to distinguish accurately .

What validation methods should I use to confirm SH3PXD2A antibody specificity?

To validate SH3PXD2A antibody specificity:

• Genetic knockdown/knockout: Use siRNA, shRNA, or CRISPR-Cas9 to create SH3PXD2A-depleted samples as negative controls

• Multiple antibodies: Compare results using antibodies raised against different epitopes of SH3PXD2A

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specific binding

• Cross-species reactivity: Test antibody performance in samples from different species where sequence homology is known

• Positive controls: Include samples with confirmed SH3PXD2A expression (e.g., HeLa or HepG2 cells)

• Blocking peptide validation: Use available blocking peptides to confirm specificity of binding

• Orthogonal techniques: Validate findings using complementary approaches (e.g., mass spectrometry, RNA expression)

For example, the PA5-58168 antibody has been tested for sequence identity with mouse (90%) and rat (89%) orthologs, providing information about expected cross-reactivity .

What are the recommended storage and handling protocols for SH3PXD2A antibodies?

For optimal antibody performance and longevity:

• Storage temperature: Store at -20°C for long-term storage

• Short-term storage: Some antibodies can be stored at 4°C for 6 months after reconstitution

• Aliquoting: Create small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality

• Buffer composition: Most commercial SH3PXD2A antibodies are supplied in PBS with glycerol (40-50%) and may contain sodium azide (0.02%) as a preservative

• Thawing procedure: Thaw antibodies completely at room temperature before use, but keep on ice for dilution

• Working dilution preparation: Dilute in fresh buffer immediately before use rather than storing diluted antibody

• Shelf life: Most antibodies are stable for 12 months from date of receipt when stored properly

Following these guidelines will help maintain antibody activity and ensure reproducible results across experiments.

How should I design experiments to study SH3PXD2A's role in cancer cell invasion?

When investigating SH3PXD2A in cancer invasion:

• Cell models: Select appropriate cell lines with varying invasive potential (HeLa and HepG2 cells express detectable SH3PXD2A levels)

• Tissue samples: Include both normal and cancerous tissues, particularly breast cancer specimens which have been validated for SH3PXD2A expression

• Functional assays: Combine antibody detection with invasion assays (Matrigel, Transwell) to correlate expression with function

• Localization studies: Use immunofluorescence to examine SH3PXD2A distribution at invasive structures

• Protein interactions: Investigate interactions with known binding partners (ADAMs, NOXs) using co-immunoprecipitation

• Signaling pathway analysis: Examine the relationship between SH3PXD2A and regulatory pathways using phospho-specific antibodies

• In vivo models: Consider using xenograft models with SH3PXD2A modulation to assess metastatic potential

Researchers have reported associations between SH3PXD2A expression and cancer progression in multiple studies .

What considerations are important when comparing results from different SH3PXD2A antibodies?

When comparing results across different antibodies:

• Epitope differences: Review immunogen sequences to understand which regions of SH3PXD2A each antibody targets

• Validation status: Check whether each antibody has been validated for your specific application and sample type

• Lot-to-lot variation: Be aware that different lots of the same antibody may show performance variations

• Cross-reactivity: Consider potential cross-reactivity with related proteins like SH3PXD2B

• Detection methods: Standardize secondary antibodies and detection systems when comparing multiple primary antibodies

• Data normalization: Use consistent loading controls and normalization methods for quantitative comparisons

• Experimental conditions: Maintain identical experimental conditions (buffers, incubation times, temperatures)

For example, antibodies targeting different epitopes (N-terminal vs. C-terminal) may yield different results based on protein folding, accessibility, or post-translational modifications.