TERF2 Antibody

What is TERF2 and why is it significant in cellular biology research?

TERF2 (also known as TRF2) is a 542 amino acid protein containing an HTH myb-type DNA-binding domain that localizes primarily in the nucleus. It serves as a critical component of the shelterin complex (telosome) that binds to telomeric double-stranded 5'-TTAGGG-3' repeats .

TERF2's biological significance stems from its central role in:

• Telomere maintenance and protection against chromosome end-to-end fusion

• Recruiting factors required for telomere protection, including TERF2IP/RAP1 and DCLRE1B/Apollo

• Telomeric loop (T-loop) formation by generating 3' single-stranded overhangs at leading end telomeres

• Regulating telomere topology during replication by controlling DNA topoisomerase activity (TOP1, TOP2A, TOP2B)

Beyond telomeric functions, recent research has revealed TERF2's involvement in:

• Regulating gene expression through binding to interstitial telomeric sequences

• Modulating immune responses and angiogenesis

• Controlling microRNA expression

• Regulating macroautophagy/autophagy through interaction with HMGB1

What applications can TERF2 antibodies be used for in laboratory research?

TERF2 antibodies have been validated for multiple experimental applications:

Research applications include studying:

• Telomere maintenance mechanisms

• Cancer progression (TERF2 is overexpressed in multiple cancer types)

• Immune cell infiltration in tumors

• Protein-protein interactions (particularly with telomeric proteins)

• Autophagy regulation

What are the molecular characteristics and reactivity profiles of commonly available TERF2 antibodies?

Commercial TERF2 antibodies exhibit the following characteristics:

When selecting an antibody for specific applications, researchers should consider reactivity demonstrated in:

• Cell lines: HeLa, MCF-7, NIH/3T3, Jurkat, K-562, HEK-293, Daudi, HSC-T6, 4T1

• Tissues: Human gliomas, rat brain, mouse brain

How should TERF2 antibodies be stored and handled for optimal performance?

For maximum stability and efficacy:

Storage Conditions:

• Store at -20°C (most commercial antibodies remain stable for one year after shipment)

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (typically smaller 20μl sizes) contain 0.1% BSA as a stabilizer

Storage Buffer Composition:

• Typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Avoid repeated freeze-thaw cycles

Handling Guidelines:

• Allow antibody to equilibrate to room temperature before opening

• For dilution, use appropriate buffer recommended for each application

• For IHC applications with TERF2 antibodies, antigen retrieval is crucial:

  • Preferred method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

Working Dilution Preparation:

• Prepare fresh working dilutions on the day of the experiment

• Discard any unused diluted antibody

What are the optimized protocols for TERF2 antibody applications in Western Blot and Immunohistochemistry?

Western Blot Protocol for TERF2 Detection:

• Sample Preparation:

  • Lyse cells in appropriate buffer containing protease inhibitors

  • Validated cell lines: HeLa, MCF-7, NIH/3T3, Jurkat, K-562

• Electrophoresis and Transfer:

  • Load 20-40 μg protein per lane

  • Expected molecular weight: 65-69 kDa (observed range)

• Blocking and Antibody Incubation:

  • Block membrane in 5% non-fat milk or BSA for 1 hour at room temperature

  • Incubate with primary TERF2 antibody at recommended dilution (1:2000-1:12000)

  • Use secondary antibody appropriate for host species

• Detection Considerations:

  • TERF2 may show multiple bands due to isoforms or processing (major bands at 65-69 kDa; minor bands at 32-35 kDa have been reported)

Immunohistochemistry Protocol for TERF2:

• Tissue Preparation:

  • FFPE sections (4-5 μm thickness)

  • Validated tissues: human gliomas, rat brain, mouse brain

• Antigen Retrieval (Critical Step):

  • Primary method: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced retrieval (pressure cooker or microwave)

• Staining Procedure:

  • Block endogenous peroxidase with peroxidase blocking solution

  • Block non-specific binding with protein blocking buffer (20 min)

  • Incubate with primary TERF2 antibody (1:50-1:500 dilution) for 1 hour at room temperature

  • Use appropriate detection system (e.g., Dako EnVision™ FLEX/HRP)

  • Develop with DAB and counterstain with Mayer's Hematoxylin

• Analysis Guidelines:

  • Score staining by combining intensity and extent scores (0-12 scale)

  • Consider scores 0-4 as negative and 5-12 as positive

  • Have two independent pathologists assess the staining

How can researchers distinguish between different forms or post-translational modifications of TERF2?

Distinguishing between TERF2 variants requires specific methodological approaches:

• Full-Length vs. Truncated Forms:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Compare with wild-type (pTERF2) and mutant (pTERF2ΔC) controls

  • Expected molecular weights:

    • Full-length TERF2: 65-69 kDa

    • Truncated forms: Various, including 32-35 kDa bands

• Post-Translational Modifications:

  • Phosphorylation: Use phospho-specific antibodies or treat samples with phosphatase

  • Ubiquitination: Use immunoprecipitation followed by ubiquitin-specific antibodies

  • Acetylation: Employ acetylation-specific antibodies

• Domain-Specific Analysis:

  • HTH myb-type DNA-binding domain can be specifically targeted

  • For functional studies, compare wild-type TERF2 with domain-deletion mutants (e.g., TERF2ΔC)

• Research Application Example:
  In autophagy studies, researchers distinguished TERF2's functional forms by:

  • Transfecting cells with wild-type (pTERF2) and mutant (pTERF2ΔC) forms

  • Using JetPEI reagent for 48h transfection

  • Employing pBabe-puro empty vector as control

What positive and negative controls should be included when working with TERF2 antibodies?

Positive Controls:

• Cell Lines with Known TERF2 Expression:

  • Human: HeLa, MCF-7, Jurkat, K-562

  • Mouse: NIH/3T3, 4T1

  • Rat: HSC-T6

• Tissue Sections:

  • Human gliomas

  • Rat brain tissue

  • Mouse brain tissue

• Genetic Controls:

  • Overexpression systems: pBabe-puro-MYC-TERF2 transfected cells

  • For functional studies: Include both wild-type TERF2 and mutant TERF2ΔC

Negative Controls:

• Antibody Controls:

  • Isotype-matched irrelevant antibody (same concentration)

  • Primary antibody omission

• Expression Controls:

  • TERF2 knockdown/silenced cells:

    • siRNA approach: Use siTERF2 (Dharmacon, 5'-GCAGAAGUGGACUGUAGAAUU-3')

    • shRNA approach: shTERF2 stable cell lines

  • TERF2 knockout cells (if available)

• Application-Specific Controls:

  • For IP experiments: Use non-immune IgG from same species

  • For ChIP: Include IgG control and primers for non-bound regions

  • For dual labeling: Single antibody controls

Technical Validation:

• Cross-reactivity assessment: Test on samples known to lack TERF2

• Peptide competition assay: Pre-incubate antibody with immunizing peptide

• Antibody titration: Perform dilution series to establish optimal concentration

How do cancer-related mutations or expression changes impact TERF2 antibody detection?

TERF2 expression and mutations vary across cancer types, affecting antibody-based detection:

• Altered Expression Patterns:

  • TERF2 is upregulated in multiple cancers including:

    • Cholangiocarcinoma (CHOL)

    • Diffuse large B-cell lymphoma (DLBC)

    • Pancreatic adenocarcinoma (PAAD)

    • Stomach adenocarcinoma (STAD)

    • Thymoma (THYM)

  • Expression correlates with tumor progression in specific cancers

• Detection Challenges in Cancer Tissues:

  • Heterogeneous expression requires careful interpretation

  • Higher antibody concentrations may be needed for low-expression regions

  • Background staining must be distinguished from specific signal

• Mutation Impact on Epitope Recognition:

  • Amplification and mutations in TERF2 particularly affect liver hepatocellular carcinoma (LIHC)

  • Mutations may alter antibody binding if they occur within the epitope region

  • Use multiple antibodies targeting different regions to verify results

• Technical Considerations for Cancer Samples:

  • For IHC scoring in cancer tissues:

    • Use immunoreactive score (IRS) combining staining intensity and percentage of positive cells

    • Apply consistent criteria: negative (scores 0-4) and positive (scores 5-12)

  • For WB analysis:

    • Include stage-matched samples when comparing expression

    • Account for tumor heterogeneity through multiple sampling

How can TERF2 antibodies be optimally employed to study telomere maintenance and dysfunction?

TERF2 antibodies are valuable tools for investigating telomere biology through advanced methodological approaches:

• Chromatin Immunoprecipitation (ChIP) for Telomere Analysis:

  • Crosslink proteins to DNA using formaldehyde (1%, 10 min)

  • Sonicate chromatin to ~500 bp fragments

  • Immunoprecipitate with TERF2 antibody

  • Analyze by qPCR using telomere-specific primers or dot blot with telomeric probe

  • Include input and IgG controls

• Co-Immunoprecipitation for Shelterin Complex Analysis:

  • Lyse cells in non-denaturing buffer to preserve protein interactions

  • Immunoprecipitate with TERF2 antibody

  • Western blot for interacting partners (TRF1, POT1, TIN2, TPP1, RAP1)

  • Validate interactions with reverse IP

  • Recommended IP conditions: 0.5-4.0 μg antibody for 1.0-3.0 mg total protein

• Immunofluorescence for Telomere Dysfunction-Induced Foci (TIF):

  • Co-stain for TERF2 and DNA damage markers (γ-H2AX, 53BP1)

  • Protocol example:

    • Fix cells in 4% formaldehyde (10 min, RT)

    • Permeabilize with 0.25% Triton X-100 (5 min, RT)

    • Incubate with rabbit anti-TERF2 and mouse anti-γ-H2AX antibodies

    • Use appropriate secondary antibodies (Alexa Fluor 488/555 conjugates)

    • Counterstain nuclei with DAPI

  • Quantify co-localization as indicator of telomere dysfunction

• Telomere Dysfunction Analysis in Cancer Models:

  • Implement TERF2 knockdown approaches:

    • Transient: siTERF2 (100 nM, 24h with INTERFERin reagent)

    • Stable: shTERF2 cell lines or conditional knockout models

  • Assess telomere protection by measuring:

    • End-to-end chromosome fusions

    • Telomere length (Q-FISH, TRF analysis)

    • ATM pathway activation

What methodological considerations are critical when investigating TERF2's role in cancer?

TERF2's complex role in tumorigenesis requires sophisticated research approaches:

• Expression Analysis Across Cancer Types:

  • Pan-cancer analysis reveals variable expression patterns:

    • Upregulated in: CHOL, DLBC, PAAD, STAD, THYM

    • Expression correlates with survival in specific cancers:

      • Poor OS in ACC, LUSC, and UVM

      • Better OS in CHOL and KIRC

  • Methodology:

    • Compare matched tumor/normal samples

    • Stratify by cancer stage and subtype

    • Correlate with clinical outcomes

• TERF2 Knockdown/Overexpression in Cancer Models:

  • For knockdown studies:

    • siRNA approach: siTERF2 (Dharmacon, 5'-GCAGAAGUGGACUGUAGAAUU-3')

    • shRNA approach for stable knockdown

  • For overexpression studies:

    • Transfect with pBabe-puro-MYC-TERF2 (wild-type) or pBabe-puro-MYC-TERF2ΔC (mutant)

    • Assess proliferation, migration, invasion, and chemosensitivity

• In Vivo Cancer Model Analysis:

  • Orthotopic injection model:

    • NSG mice injected with control (shSCR) and TERF2-silenced (shTERF2) cancer cells

    • Tumor volume monitoring

    • IHC analysis of extracted tumors with anti-TERF2 antibodies

  • Markers to assess in tumor samples:

    • Proliferation markers

    • DNA damage indicators (8-OHdG)

    • Autophagy markers (LC3B)

• TERF2 and Tumor Immune Infiltration Analysis:

  • IHC multiplex staining for TERF2 and immune cell markers

  • Correlation analysis between TERF2 expression and:

    • Cancer-associated fibroblasts (positive correlation in BLCA, CESC, HNSC, PAAD, SKCM, STAD)

    • Lymphocyte infiltration (negative correlation in GBM, LGG, UCS)

  • Chemokine expression analysis:

    • TERF2 negatively correlates with CCL2, CCL4, CCL14, CCL15, CCL28, CXCL2, CXCL3, CXCL9, CXCL16

How can researchers effectively investigate TERF2's non-canonical functions using antibody-based approaches?

Recent research has revealed TERF2's involvement in non-telomeric functions, requiring specialized methodological approaches:

• TERF2-HMGB1 Interaction in Autophagy Regulation:

  • Co-immunoprecipitation protocol:

    • Lyse cells in non-denaturing buffer

    • Immunoprecipitate with anti-TERF2 or anti-HMGB1 antibodies

    • Western blot to detect interacting partner

  • Co-localization analysis:

    • Co-immunofluorescence using rabbit anti-TERF2 and mouse anti-HMGB1 antibodies

    • Secondary antibodies: Alexa Fluor 488 (anti-rabbit) and Alexa Fluor 555 (anti-mouse)

    • Nuclear counterstain with DAPI

• TERF2's Role in Autophagy Assessment:

  • TERF2 silencing approach:

    • Transfect with siTERF2 (100 nM, 24h)

    • Autophagy stimulation: EBSS nutrient starvation

  • Autophagy markers analysis:

    • Transfect with autophagy reporters (EGFP-LC3B, mCherry-LC3B, mRFP-EGFP-LC3B)

    • Assess HMGB1 cytosolic translocation with:

      • EGFP-HMGB1 or EGFP-HMGB1 C106S constructs

      • Subcellular fractionation and western blotting

• Interstitial Telomeric Sequence Binding:

  • ChIP-seq methodology:

    • Immunoprecipitate with TERF2 antibody

    • Sequence and analyze for non-telomeric binding sites

    • Validate with ChIP-qPCR for selected targets

  • Gene expression analysis:

    • Compare expression profiles in TERF2-depleted vs. control cells

    • Correlate with TERF2 binding at specific loci

• TERF2 and microRNA Regulation:

  • RNA immunoprecipitation (RIP):

    • Crosslink RNA-protein complexes

    • Immunoprecipitate with TERF2 antibody

    • Extract and analyze associated RNAs

  • miRNA expression analysis:

    • Compare miRNA profiles in TERF2-manipulated cells

    • Validate key targets with qRT-PCR

What are the technical challenges and solutions in studying TERF2-protein interactions?

TERF2's interactions with multiple proteins present specific technical challenges:

• Preservation of Native Interactions:

  • Challenge: TERF2 interactions may be disrupted during extraction

  • Solution:

    • Use gentle lysis buffers (low detergent concentration)

    • Employ in situ proximity ligation assay (PLA) for direct visualization

    • Consider crosslinking approaches for transient interactions

• Nuclear Protein Extraction Efficiency:

  • Challenge: TERF2 is predominantly nuclear, requiring efficient nuclear extraction

  • Solution:

    • Optimize nuclear extraction protocol

    • Verify extraction efficiency with nuclear markers

    • Include DNase treatment to release DNA-bound proteins

• Distinguishing Direct vs. Indirect Interactions:

  • Challenge: Co-IP may detect both direct and indirect interactions

  • Solution:

    • Perform reciprocal IPs

    • Use domain-specific mutants (e.g., TERF2ΔC)

    • Employ in vitro binding assays with purified proteins

• Domain-Specific Interaction Mapping:

  • Challenge: Identifying interaction domains requires domain-specific tools

  • Solution:

    • Use domain-specific antibodies where available

    • Express tagged domain constructs

    • Compare interaction profiles between wild-type and mutant TERF2:

      • Wild-type: pBabe-puro-MYC-TERF2

      • Mutant: pBabe-puro-MYC-TERF2ΔC

• Validation of Novel Interactions:

  • Challenge: Confirming biological relevance of detected interactions

  • Solution:

    • Functional validation through knockdown/overexpression

    • Subcellular co-localization studies

    • Mutational analysis of interaction interfaces

How can researchers methodically investigate TERF2's role in regulating immune cell function and infiltration?

The emerging role of TERF2 in immune regulation requires specialized approaches:

• Correlation Analysis of TERF2 and Immune Cell Markers:

  • Methodology:

    • Use TIMER, XCELL, MCPCOUNTER, and EPIC algorithms

    • Correlate TERF2 expression with infiltrating immune cell populations

    • Cancer types showing significant correlations:

      • Cancer-associated fibroblasts: BLCA, CESC, HNSC, PAAD, SKCM, STAD

      • Lymphocytes: GBM, LGG, UCS (negative correlation)

• Multiparametric Analysis of Immune Markers:

  • Assess correlation between TERF2 and immune markers in tumor samples (Table from search result 5):

  Immune Cell Type	Marker	Correlation with TERF2 in ESCA Tumor	Correlation in Normal Tissue
  T cells (CD8+)	CD8A, CD8B	No significant correlation	Positive in normal tissue (GETX)
  T cells (general)	CD2, CD3E	No significant correlation	Positive in normal tissue
  Natural Killer cells	KIR2DL4, KIR3DL2	No significant correlation	Positive in normal tissue
  Th1 cells	T-bet	No significant correlation	Positive
  Th2 cells	STAT5A, STAT6	No significant correlation	Strongly positive (p<0.01)
  Tfh cells	BCL6	Positive (p<0.01)	Strongly positive (p<0.01)
  Th17 cells	STAT3	Strongly positive (p<1e-6)	Strongly positive (p<1e-4)
  T cell exhaustion	PD-1, LAG3, TIM-3	No significant correlation	Positive for TIM-3 (p<0.05)

• Chemokine Correlation Analysis:

  • Methodology:

    • Use TISIDB database

    • Analyze association between TERF2 and chemokine expression

    • Significant negative correlations with:

      • CCL2, CCL4, CCL14, CCL15, CCL28

      • CXCL2, CXCL3, CXCL9, CXCL16

• Experimental Validation in Immune Contexts:

  • In vitro approaches:

    • Co-culture TERF2-manipulated cancer cells with immune cells

    • Measure immune activation markers and effector functions

    • Assess cytokine/chemokine production

  • In vivo approaches:

    • Immunocompetent mouse models with TERF2-manipulated tumors

    • IHC analysis of tumor microenvironment

    • Flow cytometry of tumor-infiltrating immune cells

What are common issues in TERF2 antibody applications and their solutions?

How can researchers address discrepancies in TERF2 detection between different experimental systems?

Methodological approaches to resolve inconsistencies:

• Between antibody clones:

  • Test multiple antibodies targeting different epitopes

  • Compare monoclonal (for specificity) and polyclonal (for sensitivity) options

  • Verify with genetic validation (siRNA knockdown)

• Between detection methods:

  • When WB and IHC results differ:

    • Consider protein conformation differences (native vs. denatured)

    • Optimize antigen retrieval for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

    • Validate with alternative methods (IF, flow cytometry)

• Between cell/tissue types:

  • Adjust protocols for specific sample types:

    • Cell lines: Optimize lysis conditions

    • FFPE tissues: Extend antigen retrieval time

    • Fresh tissues: Adjust fixation parameters

  • Consider tissue-specific expression levels and adjust antibody concentration

• Between in vitro and in vivo results:

  • Account for microenvironment influences

  • Compare primary cells with established cell lines

  • Validate findings across multiple experimental models

What emerging technologies are enhancing TERF2 detection and functional analysis?

Advanced technological approaches:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) for multi-parameter analysis

  • Imaging mass cytometry for spatial protein detection

  • Single-cell Western blotting for heterogeneity assessment

• Super-resolution microscopy:

  • Visualize TERF2 localization at telomeres with nanometer precision

  • Techniques include STORM, PALM, and STED microscopy

  • Enables co-localization studies with other telomeric proteins

• Proximity-based protein interaction detection:

  • BioID or APEX2 proximity labeling

  • FRET/BRET for real-time interaction monitoring

  • In situ proximity ligation assay for visualizing interactions

• CRISPR-based approaches:

  • Endogenous tagging of TERF2 for live-cell imaging

  • CUT&RUN or CUT&Tag for improved chromatin binding profile

  • CRISPR interference/activation for functional studies

• Combinatorial approaches for cancer studies:

  • Multiplex IHC for simultaneous detection of TERF2 and immune markers

  • Spatial transcriptomics combined with protein detection

  • Patient-derived organoids for personalized functional studies