TLN2 Antibody

What is TLN2 and what is its biological significance?

Talin 2 (TLN2) is a large adaptor protein that links the integrin family of adhesion molecules to F-actin in the cytoskeleton. It functions as a critical component of the extracellular matrix (ECM) and can bind to various adhesion molecules including integrin, actin, and adhesion kinase . In vertebrates, there are two talin genes, TLN1 and TLN2, with TLN2 showing 74% sequence homology with TLN1 .

TLN2 expression is more tissue-restricted than TLN1, with highest expression levels in heart, brain, and skeletal muscle . Within skeletal muscle, TLN2 has been localized to both myotendinous junctions and costameres, while in cardiac tissue, TLN2 is found in costameres and intercalated discs . This distinct localization pattern suggests specialized functions for TLN2 in muscle and neural tissues. TLN2 is also reportedly the most abundant talin isoform in brain, where it is found in synapses and may participate in clathrin-mediated endocytosis through its interaction with PIP-kinase type 1γ .

How do TLN2-specific antibodies differ from other talin antibodies?

TLN2-specific antibodies are designed to recognize epitopes unique to TLN2 without cross-reactivity to TLN1. This specificity is crucial for researchers studying the distinct functions and localizations of talin isoforms. Many commercial antibodies, such as 8d4 and TD77 from Sigma, recognize both TLN1 and TLN2 isoforms, making it difficult to differentiate between them in experimental contexts .

Isoform-specific monoclonal antibodies have been developed that can distinguish between TLN1 and TLN2. For example, the monoclonal antibodies 68E7 and 121A are specific for TLN2, while 97H6 and 93E12 recognize only TLN1 . These antibodies target different epitopes within the talin proteins:

The specificity of these antibodies has been confirmed through Western blotting against cells expressing GFP-tagged talin isoforms .

What applications are TLN2 antibodies suitable for?

TLN2 antibodies have been validated for multiple research applications, with different antibodies showing utility in different techniques. Based on the available data, TLN2 antibodies have been successfully used in:

• Western Blotting (WB): TLN2-specific antibodies like 31695-1-AP can detect TLN2 in Western blots at dilutions ranging from 1:500 to 1:1000 . Isoform-specific antibodies such as 68E7 and 121A have also been validated for Western blotting applications .

• Immunoprecipitation (IP): Isoform-specific monoclonal antibodies have been successfully used for immunoprecipitation of TLN2 from cell lysates .

• Immunofluorescence (IF): TLN2 antibodies can be used for confocal laser scanning microscopy to study the subcellular localization of TLN2. For example, in differentiated human macrophages, TLN2 antibodies were used to visualize TLN2 distribution in comparison to TLN1 .

• Immunohistochemistry (IHC): TLN2-specific antibodies have been validated for localizing TLN2 in tissue sections using both cryostat and paraffin-embedded material. This has revealed tissue-specific distribution patterns, such as TLN2 localization to both myotendinous junctions and costameres in skeletal muscle .

• ELISA: Some TLN2 antibodies, such as 31695-1-AP, have been validated for ELISA applications .

What are the optimal conditions for using TLN2 antibodies in Western blotting?

For optimal Western blotting results with TLN2 antibodies, researchers should consider the following parameters:

• Sample Preparation: TLN2 is a large protein with a calculated molecular weight of 271 kDa, though it is typically observed at approximately 250 kDa on SDS-PAGE gels . Complete protein denaturation and efficient transfer of such large proteins require special attention.

• Antibody Dilution: For the TLN2 antibody 31695-1-AP, the recommended dilution range for Western blotting is 1:500 to 1:1000 . For monoclonal antibodies like 68E7, concentrated supernatants are typically used at 1:20 dilution, or purified antibodies at 5 μg/ml .

• Detection Systems: Secondary antibodies conjugated to appropriate reporter molecules (HRP, fluorophores) should be selected based on the experimental design. For fluorescence-based detection, Alexa-488 or Alexa-594 coupled secondary antibodies have been used at 1:200 dilution .

• Positive Controls: Using lysates from cells expressing GFP-tagged TLN2 as positive controls can help validate antibody specificity and optimize protocol conditions .

• Negative Controls: Including lysates from cells where TLN2 has been knocked down via siRNA can serve as negative controls to confirm antibody specificity .

It is important to note that TLN2 antibody performance may be sample-dependent, and protocols should be titrated in each testing system to obtain optimal results .

How can I ensure specificity when studying TLN2 versus TLN1?

Ensuring specificity when studying TLN2 versus TLN1 is critical due to their high sequence homology (74%) . The following strategies can help ensure specificity:

• Use Isoform-Specific Antibodies: Select antibodies that have been rigorously validated for specificity against TLN2, such as the monoclonal antibodies 68E7 and 121A, which target specific epitopes in TLN2 that are not present in TLN1 .

• Validate Antibody Specificity: Test the antibodies against cell lysates expressing recombinant TLN1 and TLN2 (e.g., GFP-tagged constructs) to confirm that they recognize only the intended isoform .

• Include Appropriate Controls:

  • Positive controls: Tissues or cell lines known to express high levels of TLN2 (e.g., brain, heart, skeletal muscle)

  • Negative controls: Tissues or cells with TLN2 knocked down using siRNA or from knockout animals

  • Cross-validation: Compare results obtained with different antibodies targeting different epitopes of TLN2

• Complementary Approaches: Combine antibody-based detection with other techniques such as RT-PCR to detect TLN2-specific transcripts or mass spectrometry for protein identification .

• Consider Tissue-Specific Expression: Be aware that TLN2 expression varies across tissues, with highest expression in heart, brain, and skeletal muscle, which can help predict where specific signals should be strongest .

What controls should be included when working with TLN2 antibodies?

When working with TLN2 antibodies, the following controls should be included to ensure reliable and interpretable results:

• Positive Tissue/Cell Controls:

  • For Western blotting: Lysates from tissues known to express high levels of TLN2 (heart, brain, skeletal muscle)

  • For immunofluorescence: Cell lines with confirmed TLN2 expression (e.g., HEK-293T cells, HeLa cells)

• Negative Controls:

  • siRNA-mediated TLN2 knockdown samples to confirm antibody specificity

  • Tissues or cells from TLN2 knockout models, if available

  • Secondary antibody-only controls to assess non-specific binding

• Isoform Specificity Controls:

  • Parallel detection with TLN1-specific antibodies to compare distribution patterns

  • Cells expressing GFP-tagged TLN1 or TLN2 to confirm antibody specificity

• Technical Controls:

  • Loading controls for Western blots to ensure equal sample loading

  • Multiple dilutions of antibodies to establish optimal working concentrations

  • Isotype control antibodies to assess non-specific binding due to Fc receptors

• Method Validation Controls:

  • Using multiple TLN2 antibodies targeting different epitopes to cross-validate findings

  • Complementary methods such as in situ hybridization to detect TLN2 mRNA

How can TLN2 antibodies be used to study tissue-specific expression patterns?

TLN2 antibodies are valuable tools for investigating the tissue-specific expression and subcellular localization of TLN2. The following approaches have been successfully employed:

• Immunohistochemistry on Tissue Sections: TLN2-specific antibodies have been used to localize TLN2 in both cryostat and paraffin-embedded tissue sections. In skeletal muscle, TLN2 was found at both myotendinous junctions and costameres, while in kidney, TLN2 co-localized with TLN1 in the glomerulus and tubular epithelial and interstitial cells of the cortex and medulla .

• Comparative Isoform Localization: Using both TLN1 and TLN2 antibodies on the same tissue sections enables researchers to compare and contrast the distribution of the two isoforms. This approach revealed that in skeletal muscle, TLN2 is present in both myotendinous junctions and costameres while TLN1 is restricted to myotendinous junctions .

• Quantitative Analysis: For quantification of TLN2-positive structures, epifluorescence microscopy images can be analyzed using software like ImageJ. After conversion to 8-bit images, thresholding techniques can be applied to determine the relative abundance of TLN2 in different cellular compartments .

• High-Resolution Imaging: Confocal laser scanning microscopy with TLN2 antibodies allows for detailed visualization of TLN2 subcellular localization. Systems such as the Leica TCS SP5 or Olympus FV1000 have been successfully used for this purpose .

• Correlation with Function: Combining TLN2 localization studies with functional assays, such as matrix degradation assays in macrophages, can reveal the functional significance of TLN2 in specific tissues .

How can TLN2 knockdown experiments be combined with antibody detection?

Combining TLN2 knockdown with antibody detection provides powerful insights into TLN2 function. The following methodological approach has been validated:

• siRNA Design and Transfection:

  • Human TLN2-specific siRNAs with sequences such as 5′-GAUGUGCGAUCACCACUAU-3′ and 5′-GGACGACCCUUCCAUGUAC-3′ have been successfully used

  • Transfection can be performed using electroporation methods, such as the MicroPorator MP-100 with 100 μl tips from the NEON transfection system

  • Optimal transfection parameters: pulse voltage 1000 V, pulse width 40 ms, pulse number 2

• Verification of Knockdown Efficiency:

  • Western blotting with TLN2-specific antibodies (e.g., 68E7) can confirm the reduction in TLN2 protein levels

  • Comparison with control siRNA-treated samples (e.g., firefly luciferase siRNA) establishes baseline expression levels

• Functional Assays Post-Knockdown:

  • In human macrophages, TLN2 knockdown led to a significant reduction in podosomal matrix degradation, revealing a functional role for TLN2 in this process

  • Immunofluorescence with TLN2 antibodies post-knockdown can reveal changes in localization patterns of remaining protein or compensatory changes in TLN1 distribution

• Time Course Studies:

  • Analyzing samples at different time points after siRNA transfection (e.g., 24, 48, 72 hours) can provide insights into the dynamics of TLN2 turnover and functional consequences of its absence

  • TLN2 antibody detection throughout this time course can quantify knockdown efficiency and recovery

• Rescue Experiments:

  • Following knockdown, transfection with siRNA-resistant TLN2 constructs and subsequent antibody detection can confirm that observed phenotypes are specifically due to TLN2 depletion

What are the challenges in detecting different TLN2 transcript variants?

Detecting different TLN2 transcript variants presents several challenges that researchers should be aware of:

• Complex Gene Structure: The TLN2 gene is much larger (~414 kb) than previously thought, with multiple 5′-exons spanning 236 kb. This complex structure generates multiple transcripts through alternative splicing .

• Tissue-Specific Expression Patterns: TLN2 transcripts vary in size from 7 to 10 kb in most tissues, with smaller transcripts detected in testis (4.8 kb) and kidney (3.9 kb). These tissue-specific expression patterns require careful selection of detection methods .

• Alternative Promoters: TLN2 has multiple promoters, including a housekeeping promoter associated with a CpG island that lacks a TATA-box. This variability in transcription initiation sites complicates the detection of 5′ ends of transcripts .

• Antibody Recognition Limitations: Antibodies targeting TLN2 protein may not distinguish between protein products of different transcript variants unless they specifically target regions affected by alternative splicing.

• Methodological Approaches:

  • RT-PCR with primers spanning different exon junctions can help identify specific transcript variants

  • 5′-RACE (Rapid Amplification of cDNA Ends) has been successfully used to identify the transcription start sites of TLN2 transcripts in different tissues

  • Northern blotting can distinguish transcript variants based on size but requires substantial amounts of RNA

  • RNA-Seq analysis can provide comprehensive identification of splice variants but requires complex bioinformatic analysis

What are common issues when using TLN2 antibodies and how can they be resolved?

Researchers may encounter various challenges when working with TLN2 antibodies. Here are common issues and strategies to address them:

• Cross-Reactivity with TLN1:

  • Issue: Many commercial antibodies recognize both TLN1 and TLN2 due to their 74% sequence homology .

  • Solution: Use rigorously validated isoform-specific antibodies like 68E7 and 121A that target unique epitopes in TLN2 . Validate specificity using cells expressing GFP-tagged TLN1 or TLN2 .

• Weak Signal in Western Blots:

  • Issue: TLN2 is a large protein (271 kDa calculated, 250 kDa observed) that may transfer inefficiently during Western blotting .

  • Solution: Optimize protein transfer conditions for large proteins (longer transfer times, lower methanol content in transfer buffer). Use the recommended antibody dilutions (1:500-1:1000 for 31695-1-AP) and titrate if necessary.

• Variable Staining Patterns in Immunofluorescence:

  • Issue: TLN2 localization may vary between cell types and tissues, complicating interpretation.

  • Solution: Include positive control tissues known to express TLN2 (heart, brain, skeletal muscle) . Compare with TLN1 staining patterns as an internal reference. Use confocal microscopy for higher resolution imaging .

• Inconsistent Results Between Experiments:

  • Issue: Variability in TLN2 antibody performance between experiments.

  • Solution: Standardize protocols, including fixation methods, antibody incubation times, and detection systems. Store antibodies according to manufacturer recommendations (e.g., -20°C with 0.02% sodium azide and 50% glycerol for 31695-1-AP) .

• Background Signal:

  • Issue: Non-specific binding leading to high background.

  • Solution: Optimize blocking conditions. Include appropriate controls (secondary antibody only, isotype controls). Consider using purified antibodies instead of supernatants when available .

How should conflicting TLN2 antibody data be interpreted?

When faced with conflicting data from different TLN2 antibodies, consider the following interpretation framework:

• Antibody Characteristics:

  • Different epitopes: Antibodies targeting different regions of TLN2 may give different results if the epitopes have differential accessibility or are affected by protein interactions or conformational changes

  • Antibody class and isotype: Compare results between polyclonal (e.g., 31695-1-AP) and monoclonal (e.g., 68E7, 121A) antibodies

• Experimental Conditions:

  • Sample preparation methods may affect epitope availability

  • Fixation protocols for immunofluorescence can impact antigen accessibility

  • Buffer conditions in Western blotting may influence antibody binding

• Validation Approaches:

  • Validate key findings with multiple antibodies targeting different epitopes

  • Complement antibody-based detection with transcript analysis

  • Use genetic approaches (siRNA knockdown, CRISPR-Cas9) to verify specificity

• Tissue and Cell Type Considerations:

  • Expression levels of TLN2 vary between tissues (higher in heart, brain, skeletal muscle)

  • Alternative splicing may affect epitope presence in different tissues

  • Post-translational modifications might mask certain epitopes in a cell type-specific manner

• Resolution Framework:

  • Systematically compare antibodies under identical conditions

  • Consider using genetic tools (TLN2 knockout or knockdown) as definitive controls

  • Employ orthogonal techniques (mass spectrometry, RNA-seq) to resolve conflicts

What factors affect TLN2 detection in different experimental contexts?

Several factors can influence the detection of TLN2 across different experimental contexts:

• Tissue-Specific Expression Levels:

  • TLN2 is more abundant in heart, brain, and skeletal muscle compared to other tissues

  • The presence of tissue-specific transcript variants may affect protein detection if antibodies target regions affected by alternative splicing

• Protein Size and Structure:

  • TLN2 is a large protein (250-271 kDa) that may require optimized protocols for efficient extraction, separation, and transfer

  • Protein conformation may affect epitope accessibility, particularly for antibodies targeting structural domains

• Experimental Techniques:

  • Western Blotting: Transfer efficiency of large proteins, buffer composition, antibody dilution (1:500-1:1000 recommended for 31695-1-AP)

  • Immunofluorescence: Fixation method, permeabilization protocol, antibody concentration (5 μg/ml for purified antibodies or 1:20 for supernatants)

  • Immunohistochemistry: Tissue processing (cryostat vs. paraffin embedding), antigen retrieval methods

• Protein Interactions:

  • TLN2 interactions with binding partners (e.g., integrin, actin, adhesion kinase) may mask certain epitopes

  • Subcellular localization varies (e.g., costameres and intercalated discs in cardiomyocytes, myotendinous junctions in skeletal muscle)

• Technical Variables:

  • Storage conditions of antibodies (recommended: -20°C with 0.02% sodium azide and 50% glycerol pH 7.3)

  • Sample handling and preparation protocols

  • Detection systems (chemiluminescence vs. fluorescence-based detection)

Understanding these factors is essential for optimizing TLN2 detection protocols and correctly interpreting experimental results across different contexts.