LPXN Antibody

What is Leupaxin (LPXN) and what cellular functions has it been implicated in?

Leupaxin (LPXN) is a member of the paxillin family of proteins with a calculated molecular weight of approximately 43 kDa, though it typically appears at around 45 kDa in experimental conditions . It serves multiple cellular functions as evidenced by current research. LPXN acts as a transcriptional coactivator for both androgen receptor (AR) and serum response factor (SRF) . Importantly, it contributes to the regulation of cell adhesion, spreading, and migration while functioning as a negative regulator in integrin-mediated cell adhesion events . In this capacity, LPXN suppresses the integrin-induced tyrosine phosphorylation of paxillin (PXN) . LPXN has also been identified as a potential adapter protein involved in the formation of adhesion zones in osteoclasts . In immune cells, particularly B cells, LPXN serves as a negative regulator of B-cell antigen receptor (BCR) signaling . The expression of LPXN has been documented in prostate cancer cells, with its expression intensity directly linked to prostate cancer progression .

What is the structural organization of LPXN and how does it relate to its function?

LPXN exhibits a domain structure characteristic of the paxillin family, featuring four leucine-rich LD-motifs in the N-terminus and four LIM domains in the C-terminus . This structural arrangement facilitates protein-protein interactions that are critical to LPXN's cellular functions. The LIM domains likely mediate interactions with cytoskeletal components and signaling proteins, while the LD motifs are known to interact with various binding partners. Research suggests LPXN may function in cell type-specific signaling by associating with PYK2, a member of the focal adhesion kinase family . This structural organization enables LPXN to function as an adapter protein, coordinating signals between cell adhesion molecules (particularly integrins) and intracellular signaling pathways. In B cells, this adapter function appears to be integral to its role in dampening BCR signaling, potentially through recruitment of inhibitory molecules to the signaling complex .

How is LPXN expression regulated in different cell types and pathological conditions?

LPXN expression patterns vary significantly across cell types and are dynamically regulated during cellular differentiation and pathological processes. In the immune system, LPXN expression is increased in germinal center B cells compared to naïve B cells, suggesting developmental regulation during B cell maturation and activation . This upregulation may serve as a feedback mechanism to control excessive B cell activation. In cancer biology, LPXN is expressed in prostate cancer cells, and its expression intensity correlates directly with prostate cancer progression, implicating it as a potential biomarker or even driver of malignant progression . Studies have also detected LPXN expression in various cell lines including Raji (B lymphocyte), PC-3 (prostate cancer), U87 (glioblastoma), MDA-MB-453 (breast cancer), and RAW264.7 (macrophage) cells, indicating its widespread presence across diverse tissue types . The mechanisms governing LPXN upregulation in cancer contexts remain incompletely characterized but may involve transcriptional activation by cancer-associated signaling pathways or epigenetic modifications affecting the LPXN genomic locus.

What experimental applications have been validated for LPXN antibodies?

LPXN antibodies have been validated for multiple experimental applications across different research contexts. Western blotting (WB) is a primary application, with recommended dilutions typically ranging from 1:2000 to 1:10000, though this varies by antibody manufacturer and sample type . Immunoprecipitation (IP) has been validated using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate, with positive detection confirmed in PC-3 cells . Immunofluorescence (IF) and immunocytochemistry (ICC) applications have been demonstrated with dilutions ranging from 1:200 to 1:800, also with positive detection in PC-3 cells . Flow cytometry has also been validated as an application for LPXN detection, with protocols typically involving cell fixation with 4% paraformaldehyde, permeabilization, and blocking with normal serum before antibody incubation . Immunohistochemistry (IHC) applications have been cited in publications, although specific protocol details were not provided in the search results . The observed molecular weight of LPXN in these applications is consistently around 43-45 kDa, aligning with theoretical predictions based on amino acid sequence .

What are the optimal conditions for using LPXN antibodies in Western blot experiments?

Optimizing Western blot protocols for LPXN detection requires careful consideration of multiple parameters to ensure specific and robust signal detection. Based on validated protocols, SDS-PAGE should be performed using gradient gels (e.g., 5-20% or 4-12% Bis-Tris) running at approximately 70-90V for 2-3 hours . For sample preparation, 25-30 μg of protein per lane under reducing conditions is typically sufficient for detection . Following electrophoresis, proteins should be transferred to a nitrocellulose membrane at approximately 150 mA for 50-90 minutes . Blocking should be performed with 5% non-fat milk in TBS for 1.5 hours at room temperature . Primary antibody incubation conditions vary by manufacturer, but successful detection has been reported using concentrations of 0.5 μg/mL incubated overnight at 4°C . Secondary antibodies conjugated to HRP should be used at dilutions around 1:2500-1:5000 for 1.5-2 hours at room temperature . Signal development using enhanced chemiluminescence (ECL) detection systems is appropriate, with LPXN typically appearing as a specific band at approximately 43 kDa, though slight variations in observed molecular weight may occur depending on sample type and post-translational modifications .

How should researchers optimize immunofluorescence protocols when using LPXN antibodies?

Successful immunofluorescence (IF) detection of LPXN requires optimization of fixation, permeabilization, blocking, and antibody incubation parameters. For cultured cells, fixation with 4% paraformaldehyde is recommended, followed by permeabilization with an appropriate buffer containing detergents like Triton X-100 . Blocking should be performed using 10% normal serum (typically from the same species as the secondary antibody) to minimize background staining . Primary LPXN antibodies have been successfully used at dilutions of 1:200 to 1:800, though researchers should empirically determine the optimal concentration for their specific experimental setup . For optimal staining, primary antibody incubation should typically be conducted for 30 minutes to overnight at temperatures ranging from 4°C to 20°C depending on the protocol . Fluorophore-conjugated secondary antibodies (e.g., DyLight®488) can be used at concentrations of 5-10 μg per 1×10^6 cells with 30-minute incubation at 20°C . Proper controls are essential, including an isotype control antibody (e.g., rabbit IgG at equivalent concentration to the primary antibody) and unstained samples to account for any autofluorescence . PC-3 cells have been confirmed as a positive control for LPXN detection by IF, making them a suitable model system for initial protocol optimization .

How do LPXN antibodies contribute to understanding B cell biology and immune responses?

LPXN antibodies have provided valuable insights into B cell biology by enabling detection and functional analysis of this protein in immune contexts. Research utilizing these antibodies has revealed that LPXN expression is upregulated in germinal center B cells compared to naïve B cells, suggesting potential roles in B cell activation and differentiation . Studies employing LPXN antibodies in combination with Lpxn knockout models have demonstrated that while LPXN deficiency leads to decreased B cell differentiation into plasma cells in vitro, it appears dispensable for generating potent B cell immune responses in vivo . This apparent contradiction between in vitro and in vivo findings highlights the complexity of B cell signaling networks and suggests compensatory mechanisms may exist in intact organisms. LPXN antibodies have helped establish that the protein functions as a negative regulator of B-cell antigen receptor (BCR) signaling, potentially serving as a brake on excessive immune activation . Western blot analysis using anti-LPXN antibodies has confirmed expression in lymphoid cell lines like Raji, providing cellular models for investigating LPXN function in B cells . Flow cytometry applications with LPXN antibodies have permitted quantitative assessment of protein expression levels across B cell populations, facilitating studies of its regulation during immune responses .

What methodological approaches can resolve discrepancies between in vitro and in vivo LPXN function in B cells?

The observed discrepancy between LPXN's apparent importance for in vitro plasma cell differentiation versus its dispensability in vivo requires sophisticated methodological approaches to resolve. Researchers should consider employing tissue-specific and inducible knockout models that allow temporal control over LPXN deletion, potentially revealing stage-specific requirements masked in constitutive knockout systems . Single-cell analysis techniques combined with LPXN antibody staining could identify specific B cell subpopulations where LPXN function is critical, which might be obscured in bulk population studies. Phospho-specific antibodies targeting LPXN or its interaction partners could elucidate differences in signaling dynamics between in vitro and in vivo conditions. Comprehensive immunophenotyping of B cell populations from wild-type versus Lpxn-deficient mice under various immunization protocols (T-dependent and T-independent) would help characterize subtle phenotypes that might be missed in standard assays . Investigating potential compensatory mechanisms in vivo could involve proteomics approaches to identify upregulated proteins in Lpxn-deficient B cells compared to wild-type controls. Ex vivo culture systems that better recapitulate the lymphoid microenvironment might bridge the gap between simplified in vitro systems and complex in vivo models. Finally, adopting stress conditions or secondary challenges might reveal conditional requirements for LPXN function that are not apparent under standard experimental conditions.

How might LPXN antibodies be utilized to investigate potential roles in cancer biology?

LPXN antibodies represent powerful tools for investigating this protein's roles in cancer biology, particularly given evidence linking LPXN expression to prostate cancer progression . Immunohistochemical analysis of tumor tissue microarrays using validated LPXN antibodies can establish correlations between expression levels and clinical parameters including tumor grade, stage, and patient outcomes. Cell line studies employing Western blot, immunofluorescence, and flow cytometry with LPXN antibodies can characterize expression across diverse cancer types, with particular attention to prostate cancer cell lines like PC-3 where expression has been confirmed . Co-immunoprecipitation experiments using LPXN antibodies can identify cancer-specific interaction partners, potentially revealing unique signaling complexes in malignant contexts. Combining LPXN antibodies with phospho-specific antibodies in multiplex immunofluorescence or flow cytometry could reveal activation states of associated signaling pathways in cancer cells. Functional studies using LPXN knockdown or overexpression, validated by antibody-based detection methods, could establish causal relationships between LPXN levels and cancer cell behaviors including proliferation, migration, and drug resistance. Chromatin immunoprecipitation (ChIP) using LPXN antibodies might identify genomic loci directly regulated by LPXN in its capacity as a transcriptional coactivator for androgen receptor, potentially revealing cancer-relevant target genes . Finally, examining LPXN expression in circulating tumor cells using flow cytometry or immunofluorescence could explore its utility as a liquid biopsy biomarker.

Why might researchers observe variable band sizes when detecting LPXN via Western blot?

Variability in LPXN band sizes observed in Western blot experiments can stem from multiple factors that researchers should systematically address. While the calculated molecular weight of LPXN is 43 kDa, it is commonly observed at approximately 45 kDa in experimental conditions . This discrepancy itself represents a normal variation that can be attributed to post-translational modifications (PTMs) such as phosphorylation, which is particularly relevant given LPXN's role in signaling pathways. Different isoforms or splice variants of LPXN might exist across tissue types, potentially resulting in bands of varying molecular weights. Proteolytic degradation during sample preparation can generate lower molecular weight fragments, which can be minimized by including protease inhibitors in lysis buffers and maintaining samples at cold temperatures. Conversely, incomplete denaturation or reduction can result in higher molecular weight complexes, requiring optimization of sample preparation conditions including SDS concentration, reducing agent strength, and heating duration/temperature. Non-specific binding of LPXN antibodies, particularly polyclonal preparations, might detect related family members like paxillin, which shares structural similarity with LPXN . Different detection systems and image acquisition settings can affect the apparent size and resolution of bands, necessitating appropriate size standards and consistent imaging protocols. Finally, variations in gel percentage, running conditions, and buffer systems can affect protein migration patterns and apparent molecular weights.

What controls are essential when working with LPXN antibodies in immunoprecipitation experiments?

Implementing rigorous controls is critical for reliable interpretation of immunoprecipitation (IP) experiments using LPXN antibodies. An isotype control antibody of the same species and immunoglobulin class as the LPXN antibody should be used in parallel IP reactions to identify non-specific binding . A positive control sample known to express LPXN, such as PC-3 cells which have been validated for LPXN IP applications, should be included to confirm assay functionality . Pre-clearing lysates with protein A/G beads prior to adding LPXN antibody can reduce non-specific binding. Input controls (typically 5-10% of the lysate used for IP) must be analyzed alongside IP samples to assess enrichment efficiency. For assessing antibody specificity, comparing IP results from wild-type samples versus those from LPXN-knockout or knockdown models provides definitive validation. When exploring protein-protein interactions, reciprocal IP experiments should be performed where possible, using antibodies against the putative interaction partner to confirm the association. Denaturing elution conditions can help distinguish direct binding from indirect complex formation. Western blot detection of IP samples should include probing for known LPXN-interacting proteins as positive controls, while probing for non-interacting proteins serves as negative controls. For quantitative applications, spiking lysates with recombinant LPXN at known concentrations can establish a standard curve for quantification. Finally, validating results across multiple cell types or tissue sources increases confidence in the biological relevance of findings.

How can researchers distinguish between specific and non-specific binding when using LPXN antibodies?

Distinguishing specific from non-specific binding is a fundamental challenge when working with antibodies, including those targeting LPXN. Validation using samples from knockout or knockdown models provides the most definitive assessment of antibody specificity, as the target-specific signal should be absent or significantly reduced in these samples . Antibody titration experiments, testing a range of concentrations, can help identify the optimal working dilution where specific signal is maximized relative to background. Competition assays using recombinant LPXN protein can confirm specificity, as pre-incubation of the antibody with excess target protein should block specific binding. Multiple antibodies targeting different epitopes of LPXN should generate consistent results in terms of cellular/subcellular localization and molecular weight, increasing confidence in signal specificity. Comparing staining patterns or bands with published literature and antibody validation data helps confirm expected results . For immunofluorescence or flow cytometry, secondary-only controls are essential to identify background from the detection system, while isotype controls help distinguish non-specific binding of primary antibodies . Positive control samples with known LPXN expression (e.g., PC-3 cells, Raji cells) provide important benchmarks for anticipated signal patterns . Western blot analysis should include multiple cell lines/tissues with varying LPXN expression to confirm signal correlation with expected biological variation . Finally, correlating protein detection with mRNA expression data can provide additional validation, particularly when examining expression across multiple cell types or experimental conditions.

What emerging applications of LPXN antibodies may advance understanding of cell signaling networks?

LPXN antibodies have significant potential to advance our understanding of complex signaling networks through emerging research applications. Proximity ligation assays (PLA) using LPXN antibodies paired with antibodies against potential interaction partners could reveal spatiotemporal dynamics of signaling complexes at single-molecule resolution. Mass spectrometry combined with LPXN immunoprecipitation could identify novel interaction partners and post-translational modifications under various cellular conditions. Phospho-specific LPXN antibodies, though not mentioned in the search results, could be developed to monitor activation states in response to various stimuli, providing insight into signaling kinetics. Super-resolution microscopy techniques employing fluorescently-labeled LPXN antibodies could reveal nanoscale organization of adhesion complexes and signaling hubs. ChIP-seq using LPXN antibodies would map genome-wide binding sites, elucidating its function as a transcriptional coactivator for androgen receptor and serum response factor . Live-cell imaging with cell-permeable LPXN antibody fragments or nanobodies could track dynamic changes in localization during cellular processes. Single-cell western blot or mass cytometry using LPXN antibodies could characterize heterogeneity within cell populations. Finally, LPXN antibodies could be employed in therapeutic contexts, either as targeting moieties for drug delivery systems or as tools for isolating specific cell populations for personalized medicine applications.

How might new antibody technologies enhance LPXN detection and functional characterization?

Emerging antibody technologies promise to significantly enhance LPXN detection sensitivity, specificity, and functional characterization capabilities. Recombinant antibody technologies can generate highly specific monoclonal antibodies against multiple LPXN epitopes, enabling more consistent results across research laboratories. Nanobodies or single-domain antibodies derived from camelid immunoglobulins offer advantages for detecting LPXN in live cells or crowded molecular environments due to their small size. Bispecific antibodies targeting LPXN and its interaction partners could enable selective detection of specific protein complexes involved in signaling or transcriptional regulation. Antibody engineering approaches incorporating site-specific conjugation of fluorophores or other detection moieties can improve signal-to-noise ratios in various applications. Intrabodies designed to recognize and track LPXN in living cells could reveal dynamic localization patterns during cell migration, adhesion, or signaling events. Aptamer-antibody conjugates might combine the specificity of antibodies with the versatility of nucleic acid aptamers for novel detection strategies. Split-antibody complementation systems could enable real-time monitoring of LPXN interactions with binding partners. Antibody arrays and multiplexed detection platforms incorporating LPXN antibodies alongside antibodies against related signaling proteins could facilitate comprehensive pathway analysis. Finally, microfluidic antibody-based detection systems might enable analysis of LPXN in limited samples such as rare cell populations or patient biopsies.

What are the key considerations for researchers selecting LPXN antibodies for specific applications?

When selecting LPXN antibodies for research applications, investigators should consider several critical factors to ensure experimental success. Validation status for the specific intended application should be thoroughly reviewed, with preference given to antibodies that have demonstrated utility in Western blot, immunoprecipitation, immunofluorescence, or flow cytometry as required . Species reactivity must align with experimental models, noting that while human reactivity is well-documented for many commercial antibodies, cross-reactivity with mouse LPXN should be confirmed for murine studies . Antibody type considerations include trade-offs between polyclonal antibodies (offering broader epitope recognition but potential batch variability) versus monoclonal antibodies (providing consistency but potentially limited to single epitopes that might be inaccessible in certain applications). Clone selection for monoclonal antibodies should account for the target epitope location relative to functional domains or regions involved in protein-protein interactions. Host species should be selected to avoid cross-reactivity with endogenous immunoglobulins in the experimental system and to ensure compatibility with other antibodies in multiplex applications. Recommended working dilutions vary considerably between applications (1:2000-1:10000 for WB, 1:200-1:800 for IF/ICC) , necessitating empirical optimization for each experimental context. Conjugation options (unconjugated versus directly labeled) should be selected based on the detection system and experimental design. Finally, published literature citing specific antibody catalog numbers provides valuable validation and can guide selection of reliable reagents.

How will understanding LPXN biology impact future therapeutic approaches?

The evolving understanding of LPXN biology, facilitated by antibody-based research, may inform novel therapeutic strategies across multiple disease contexts. In cancer biology, particularly prostate cancer where LPXN expression correlates with disease progression, targeting this protein or its interactions might offer therapeutic opportunities . Its role as a transcriptional coactivator for androgen receptor suggests potential relevance to hormone therapy resistance mechanisms in prostate cancer . The negative regulatory function of LPXN in B-cell receptor signaling indicates possible applications in autoimmune disorders characterized by B cell hyperactivation . Although LPXN appears dispensable for normal B cell immune responses in vivo, its importance might become evident under pathological conditions or stress, warranting further investigation . The involvement of LPXN in integrin-mediated cell adhesion and migration processes suggests potential relevance to metastasis inhibition strategies . Structure-based drug design targeting specific LPXN domains or protein-protein interactions could yield selective modulators with therapeutic potential. Antibody-drug conjugates directed against LPXN might enable targeted delivery of cytotoxic agents to cancer cells expressing elevated levels of this protein. RNA interference or CRISPR-based approaches targeting LPXN could be explored as therapeutic strategies in appropriate disease contexts. Finally, the development of biomarkers based on LPXN expression or activation status could enable patient stratification for personalized medicine approaches in cancer treatment or immunotherapy.

What resources are available for validation and troubleshooting of LPXN antibodies?

Researchers working with LPXN antibodies can access various resources for validation and troubleshooting experiments. Commercial antibody providers like Proteintech, Abcam, and Boster Bio offer product-specific validation data including Western blot images, immunofluorescence staining patterns, and flow cytometry results that serve as benchmarks for expected results . Published literature cited in product documentation provides independent validation of antibody performance in specific applications, with at least one publication mentioned for immunohistochemistry and immunofluorescence applications of anti-LPXN antibodies . Detailed protocols from manufacturers specify recommended conditions for various applications, including sample preparation, antibody dilutions, incubation times/temperatures, and detection methods . Known positive control samples include PC-3 and DU 145 prostate cancer cell lines, Raji B lymphocytes, and human spleen tissue, which have been validated for LPXN expression . Antibody repositories like Antibodypedia aggregate user experiences and validation data across multiple antibodies targeting the same protein. Human Protein Atlas provides tissue and cell line expression data for LPXN at both protein and mRNA levels, offering baseline expectations for expression patterns. Cell lines derived from LPXN knockout models, though not explicitly mentioned in the search results, would provide invaluable negative controls for antibody validation. Online troubleshooting guides from antibody manufacturers address common issues in various applications. Finally, research forums and communities provide platforms for researchers to share experiences and optimization strategies when working with challenging antibodies.