TAGLN2 Antibody

What is TAGLN2 and why is it significant in immunological research?

TAGLN2 (Transgelin-2) is a 22-kDa actin-binding protein that is predominantly expressed in T cells and immune tissues. It plays crucial roles in multiple immunological processes, particularly in T cell activation and phagocytosis. TAGLN2 functions by stabilizing cortical F-actin, which maintains F-actin content at the immunological synapse (IS) following T cell receptor stimulation, enabling proper T cell activation .

Unlike other transgelin family members (TAGLN1 and TAGLN3), TAGLN2 is specifically enriched in immune tissues including the thymus, spleen, and lymph nodes. Its unique expression pattern makes it an important target for immunological research . When using TAGLN2 antibodies, researchers should be aware that while other transgelin family members may share sequence homology, their tissue distribution differs significantly, with TAGLN2 being the predominant isoform in immune cells .

What are the recommended applications for TAGLN2 antibodies in research?

TAGLN2 antibodies can be effectively utilized across multiple experimental applications:

• Western Blotting: TAGLN2 antibodies reliably detect the 22-kDa protein in cell and tissue lysates. Recommended dilutions typically range from 1:500 to 1:1000, but optimization may be required based on specific antibody characteristics .

• Immunohistochemistry (IHC): TAGLN2 antibodies have been successfully used for detecting expression in placental tissues, revealing expression in different trophoblast subtypes, including villous syncytiotrophoblasts (STBs), cytotrophoblasts (CTBs), and interstitial extravillous trophoblasts (iEVTs) .

• Immunofluorescence: For co-localization studies with actin, TAGLN2 antibodies have proven effective in demonstrating TAGLN2's association with actin stress fibers and its localization to lamellipodial leading edges .

• Immunoprecipitation: TAGLN2 antibodies can be used to investigate protein-protein interactions, as demonstrated in studies examining TAGLN2's interaction with actin, which was found to be stimulus-independent .

When selecting an antibody, researchers should consider the host species, clonality (monoclonal vs. polyclonal), and validated applications to ensure compatibility with their experimental design.

How should I optimize TAGLN2 antibody staining for immunohistochemistry?

For optimal immunohistochemical staining of TAGLN2 in tissue sections, consider the following protocol adaptations:

• Fixation: Use 4% paraformaldehyde for adequate preservation of protein structure while maintaining antigenic epitopes.

• Antigen Retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for TAGLN2 detection.

• Antibody Dilution: Start with a 1:100 dilution and optimize based on signal-to-noise ratio. Studies examining TAGLN2 in placental tissues have successfully employed this approach .

• Detection System: An avidin-biotin complex (ABC) method with 3,3′-diaminobenzidine (DAB) as chromogen has proven effective for visualizing TAGLN2 expression patterns.

• Controls: Include both positive controls (tissues known to express TAGLN2, such as spleen or thymus) and negative controls (either omitting primary antibody or using tissues from TAGLN2 knockout models) .

When interpreting IHC results, note that TAGLN2 expression may vary with tissue type and physiological state. For instance, in placental tissues, TAGLN2 expression gradually weakens as pregnancy progresses, with significantly decreased expression observed in preeclamptic placentas compared to normal placentas .

How can TAGLN2 antibodies be used to investigate mechanisms of cancer progression?

TAGLN2 antibodies serve as valuable tools for investigating cancer progression mechanisms, particularly in gastric cancer research. Studies have revealed that TAGLN2 is aberrantly upregulated in gastric cancer and contributes to chemotherapy and radiation resistance . Researchers can employ the following methodologies:

• Expression Analysis in Cancer Tissues: Utilize TAGLN2 antibodies for IHC or western blot analysis to compare expression levels between tumor and adjacent normal tissues. Correlation analyses can then be performed with clinicopathological features to assess prognostic significance.

• Signaling Pathway Investigation: TAGLN2 antibodies can help elucidate the role of TAGLN2 in activating resistance signature Interferon-stimulated genes (ISGs) through the AKT-YBX1 pathway. Specifically, TAGLN2 has been shown to modulate YBX1 by recruiting c-Myc and SOX9 to the YBX1 promoter region and directly interacting with AKT-YBX1, enhancing YBX1 phosphorylation and nuclear translocation .

• DNA Repair Mechanism Studies: When combined with γH2AX antibodies, TAGLN2 antibodies can be used to investigate the role of TAGLN2 in DNA repair processes. Research has demonstrated that TAGLN2 depletion increases γH2AX signal in the nucleus, indicating its function in DNA repair .

• Resistance Mechanism Analysis: For studies examining therapy resistance, TAGLN2 antibodies can be employed in immunoprecipitation experiments to identify protein complexes involved in resistance pathways. Gene set enrichment analysis has shown that TAGLN2 is involved in multiple pathways including DNA repair, G2/M checkpoint, and IL6-JAK-STAT3 signaling .

What methods can be used to study TAGLN2's interaction with the actin cytoskeleton?

To investigate TAGLN2's interaction with the actin cytoskeleton, researchers can employ several sophisticated approaches:

• Co-immunoprecipitation (Co-IP): Using TAGLN2 antibodies to pull down protein complexes, followed by western blotting for actin, can confirm physical interactions between TAGLN2 and actin. This technique has revealed that TAGLN2 binding to actin is stimulus-independent in T cells .

• Immunofluorescence Co-localization: Double staining with TAGLN2 antibodies and fluorescently labeled phalloidin (for F-actin) can visualize co-localization patterns. TAGLN2 has been observed to colocalize with actin stress fibers and localize to the lamellipodial leading edge where actin polymerization actively occurs .

• Live Cell Imaging with GFP-tagged TAGLN2: Combining TAGLN2 antibodies with GFP-tagged TAGLN2 constructs allows for validation of antibody specificity and dynamic visualization of TAGLN2-actin interactions in living cells.

• Mutational Analysis: Antibodies against TAGLN2 can be used to compare wild-type and mutant protein interactions with actin. Studies have shown that deletion of the actin-binding motif (ABM, residues 153-160) reduces TAGLN2 association with F-actin and eliminates its actin-stabilizing activity .

• F-actin Stabilization Assays: Researchers can employ in vitro assays combining purified TAGLN2 (detected using TAGLN2 antibodies) with fluorescently labeled actin to assess its effect on actin depolymerization. TAGLN2 has been shown to compete with cofilin, an actin-depolymerizing factor, both in vitro and in vivo .

How can TAGLN2 antibodies be used to study T cell immunological synapse formation?

TAGLN2 antibodies are instrumental in investigating immunological synapse (IS) formation in T cells through the following methodologies:

• Confocal Microscopy of the IS: TAGLN2 antibodies can be used to visualize TAGLN2 localization during IS formation. Research has shown that endogenous TAGLN2 localizes to the F-actin ring of the distal supramolecular activation cluster (d-SMAC) in the IS .

• Quantitative Analysis of IS Components: Using TAGLN2 antibodies alongside antibodies against other IS components allows for quantitative assessment of protein redistribution during T cell activation.

• Functional Analysis in TAGLN2-deficient Cells: TAGLN2 antibodies can confirm knockout efficiency in TAGLN2-deficient cells, which have been shown to display reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading .

• Cytokine Production Assays: Combined with intracellular cytokine staining, TAGLN2 antibodies can help correlate TAGLN2 expression with functional outcomes such as IL-2 production. TAGLN2 knockout has been associated with weakened cytokine production and cytotoxic effector function .

To optimize these experiments, researchers should consider using high-resolution imaging techniques such as total internal reflection fluorescence (TIRF) microscopy or structured illumination microscopy (SIM) for detailed visualization of TAGLN2 distribution at the IS.

How can I address non-specific binding when using TAGLN2 antibodies?

Non-specific binding is a common challenge when working with antibodies. For TAGLN2 antibodies, researchers can implement the following troubleshooting strategies:

• Antibody Titration: Determine the optimal antibody concentration that provides specific signal while minimizing background. Start with manufacturer recommendations and perform serial dilutions.

• Blocking Optimization: Extend blocking time (1-2 hours at room temperature) with 5% BSA or 5-10% normal serum from the same species as the secondary antibody.

• Validation in Knockout Models: Include samples from TAGLN2 knockout models as negative controls to definitively identify non-specific binding. Studies have utilized TAGLN2−/− tissues to confirm antibody specificity .

• Cross-Reactivity Assessment: Test for cross-reactivity with other transgelin family members (TAGLN1, TAGLN3) in tissues where these proteins are highly expressed (smooth muscle for TAGLN1, neurons for TAGLN3). This is particularly important as TAGLN2 shares sequence homology with these family members .

• Alternative Antibody Clones: If persistent non-specific binding occurs, try antibodies that recognize different epitopes of TAGLN2.

When publishing results, always include detailed information about antibody validation procedures to strengthen the reliability of your findings.

What controls should be included when studying TAGLN2 expression in different cell types?

When investigating TAGLN2 expression across different cell types, incorporate these essential controls:

• Positive Control Tissues/Cells: Include samples known to express high levels of TAGLN2, such as thymus, spleen, or lymph nodes. T cells consistently express high levels of TAGLN2 and serve as excellent positive controls .

• Negative Control Tissues/Cells: Include samples with negligible TAGLN2 expression. Based on tissue expression profiles, certain non-lymphoid tissues may serve this purpose.

• siRNA Knockdown Controls: Cells treated with TAGLN2-specific siRNA provide valuable specificity controls. The efficiency of TAGLN2 knockdown should be confirmed by western blot analysis .

• Isotype Controls: Include appropriate isotype controls matching the primary TAGLN2 antibody class and concentration to identify potential non-specific binding.

• TAGLN2 Knockout Models: When available, tissues or cells from TAGLN2−/− models provide definitive negative controls for antibody specificity validation .

What are the best practices for quantitative analysis of TAGLN2 expression?

For accurate quantitative analysis of TAGLN2 expression:

• Western Blot Quantification:

  • Use housekeeping proteins (β-actin, GAPDH) for normalization

  • Employ technical replicates (minimum n=3)

  • Utilize appropriate software (ImageJ, Image Lab) for densitometric analysis

  • Assess linearity of signal by loading protein concentration gradients

• IHC/Immunofluorescence Quantification:

  • Use consistent exposure settings across all samples

  • Analyze multiple fields per sample (minimum 5-10 random fields)

  • Employ appropriate software for quantifying staining intensity

  • Consider both staining intensity and percentage of positive cells

• Flow Cytometry Analysis:

  • Include fluorescence minus one (FMO) controls

  • Use median fluorescence intensity (MFI) rather than mean values

  • Analyze sufficient events (minimum 10,000 cells of interest)

• qPCR Analysis:

  • Use multiple reference genes for normalization

  • Verify primer efficiency with standard curves

  • Apply appropriate statistical methods for analyzing fold changes

When reporting results, include detailed methodological information including antibody dilutions, exposure times, and quantification parameters to ensure reproducibility.

How can TAGLN2 antibodies be used to investigate preeclampsia mechanisms?

TAGLN2 antibodies offer valuable tools for investigating preeclampsia (PE) mechanisms, as recent research has revealed decreased TAGLN2 expression in preeclamptic placentas. Researchers can implement the following approaches:

• Comparative Expression Analysis: Use TAGLN2 antibodies for IHC and western blot analysis to compare expression between normal and PE placentas. Studies have shown that TAGLN2 expression weakens as pregnancy progresses, with significantly decreased expression in PE placentas compared to normal placentas .

• Trophoblast Function Investigation: TAGLN2 antibodies can be used to study trophoblast migration, invasion, and fusion—processes critical to placentation and compromised in PE. Research has demonstrated that TAGLN2 knockdown significantly decreases the migratory and invasive abilities of trophoblast cells .

• E-cadherin Interaction Studies: TAGLN2 antibodies can be employed in co-immunoprecipitation experiments to investigate TAGLN2's interaction with E-cadherin, which appears to be a key mechanism by which TAGLN2 regulates trophoblast invasion and syncytialization .

• In Vivo Model Validation: When studying animal models of PE, TAGLN2 antibodies can confirm knockdown efficiency and examine phenotypic consequences. TAGLN2-deficient mice have exhibited PE-like phenotypes including higher systolic blood pressure and lower placental and fetal weights .

What experimental approaches can be used to study TAGLN2's role in DNA repair and interferon signaling?

To investigate TAGLN2's emerging roles in DNA repair and interferon signaling pathways, researchers can employ these sophisticated approaches:

• Subcellular Fractionation and Western Blotting: Using TAGLN2 antibodies, researchers can analyze TAGLN2 distribution in cytoplasmic, nuclear, and chromatin-bound fractions before and after DNA damage. This approach helps determine how TAGLN2 may shuttle between compartments during DNA repair processes.

• Immunofluorescence Co-localization with DNA Damage Markers: Combining TAGLN2 antibodies with antibodies against γH2AX allows visualization of TAGLN2 recruitment to DNA damage sites. Studies have shown that TAGLN2 depletion increases γH2AX signal in the nucleus, indicating its function in DNA repair .

• BrdU Incorporation Assays: TAGLN2 antibodies can be used alongside BrdU staining to investigate the role of TAGLN2 in generating cytosolic single-stranded DNA (ssDNA). Research has shown that TAGLN2 depletion markedly reduces cytosolic ssDNA accumulation .

• Chromatin Immunoprecipitation (ChIP): TAGLN2 antibodies can be used in ChIP experiments to investigate TAGLN2's potential role in transcriptional regulation of interferon-stimulated genes (ISGs). This approach could elucidate how TAGLN2 contributes to ISG upregulation.

• Pathway Analysis: Combined with phospho-specific antibodies (e.g., phospho-AKT, phospho-STAT), TAGLN2 antibodies can help map signaling cascades linking TAGLN2 to interferon responses. Gene set enrichment analysis has shown TAGLN2 involvement in IFN alpha/gamma response pathways .

How can TAGLN2 antibodies contribute to understanding phagocytosis mechanisms?

TAGLN2 antibodies can significantly advance our understanding of phagocytosis mechanisms, particularly in macrophages, through these methodologies:

• Expression Analysis in Activated Macrophages: TAGLN2 antibodies can be used to monitor TAGLN2 upregulation in macrophages following LPS stimulation. Studies have shown that TAGLN2 is greatly induced in macrophages in response to LPS, partly via the NF-κB pathway .

• Phagocytosis Functional Assays: TAGLN2 antibodies can confirm knockout or knockdown efficiency in models studying phagocytic function. TAGLN2-deficient macrophages have demonstrated defective phagocytic functions against IgM- and IgG-coated sheep red blood cells as well as bacteria .

• Signaling Pathway Investigation: Combined with phospho-specific antibodies, TAGLN2 antibodies can help elucidate signaling pathways affected by TAGLN2 deficiency. Research has shown that cell signaling pathways involved in actin rearrangement—PI3 kinase/AKT and Ras-ERK—are down-regulated in LPS-stimulated TAGLN2-deficient macrophages .

• In Vivo Infection Models: TAGLN2 antibodies can be used to assess TAGLN2 expression in tissues from infection models. TAGLN2−/− mice have shown higher mortality after bacterial infection than wild-type littermates, suggesting TAGLN2's role as a molecular component required for host defense .

For optimal results in phagocytosis studies, researchers should consider using primary macrophages rather than cell lines and implement standardized phagocytosis assays with fluorescently labeled targets to quantify phagocytic activity precisely.

What are the emerging applications of TAGLN2 antibodies in clinical research?

While TAGLN2 research has primarily focused on basic mechanisms, emerging applications with clinical relevance include:

• Cancer Biomarker Development: TAGLN2 antibodies may be valuable for developing prognostic or predictive biomarkers, particularly in gastric cancer where TAGLN2 upregulation is associated with chemotherapy and radiation resistance .

• Preeclampsia Risk Assessment: Given the decreased expression of TAGLN2 in preeclamptic placentas, TAGLN2 antibodies could potentially contribute to the development of early diagnostic tools for preeclampsia risk .

• Immunotherapy Response Prediction: As TAGLN2 plays a crucial role in T cell activation, TAGLN2 antibodies might help predict responses to immunotherapies that depend on T cell function .

• Sepsis Prediction and Monitoring: Since TAGLN2 is essential for phagocytosis and host defense against bacteria, TAGLN2 antibodies could be useful in studying macrophage function in sepsis models .