TARBP2 Antibody

What is TARBP2 and why is it an important research target?

TARBP2 (TAR RNA-binding protein 2), also known as TRBP, is a multifunctional protein that plays crucial roles in several cellular pathways. It functions as a subunit of the RISC loading complex and contains three double-stranded RNA-binding domains (DSRBDs). The human version of TRBP has a canonical length of 366 amino acid residues and a molecular weight of approximately 39 kDa .

TARBP2 is important in research because it:

• Functions in microRNA processing and RISC assembly

• Binds to HIV-1 TAR RNA and activates HIV-1 gene expression

• Regulates RNA stability and splicing in the nucleus

• Plays a role in muscle cell differentiation

• Has been implicated in cancer progression, particularly lung cancer

• Participates in immune responses against certain viruses

TARBP2 is localized in both the nucleus and cytoplasm, with distinct functions in each compartment . This dual localization makes it an interesting target for studying various cellular processes.

What are the key considerations when selecting a TARBP2 antibody for experiments?

When selecting a TARBP2 antibody, researchers should consider:

Antibody Format and Application Compatibility:

Species Reactivity: Ensure the antibody recognizes TARBP2 from your experimental species. Many available antibodies react with human, mouse, and rat TARBP2 .

Epitope Location: Consider whether the target epitope is in a functional domain of TARBP2. For instance, antibodies targeting the dsRNA-binding domains might interfere with RNA-binding capacity in certain applications.

Validation Data: Review the validation data provided by manufacturers, including Western blot images, IHC staining patterns, and specificity tests .

Cellular Localization Studies: If studying the nuclear versus cytoplasmic functions of TARBP2, verify that the antibody can detect the protein in both compartments, as TARBP2 has been shown to localize to both areas .

How can I optimize Western blot protocols for TARBP2 detection?

For optimal Western blot detection of TARBP2, follow these research-validated recommendations:

Sample Preparation:

• Include both nuclear and cytoplasmic fractions since TARBP2 is found in both compartments

• Use protease inhibitors to prevent degradation

• Consider phosphatase inhibitors if studying post-translational modifications

SDS-PAGE Conditions:

• Use 10-12% acrylamide gels to properly resolve the ~39 kDa TARBP2 protein

• Include positive control samples (e.g., recombinant TARBP2)

Antibody Dilution and Incubation:

• For monoclonal antibodies, optimal dilutions are typically between 1:1000-1:2000

• For polyclonal antibodies, start with dilutions of 1:500-1:1000

• Most TARBP2 antibodies work well with standard blocking buffers (5% non-fat milk or BSA)

Signal Detection:

• Expect to see a band at approximately 39 kDa

• Some splice variants may produce additional bands

• TARBP2 may also show post-translational modifications affecting migration

Validation Controls:

• Use TARBP2 knockdown or knockout samples as negative controls

• Consider using recombinant TARBP2 protein as a positive control

What are the optimal conditions for immunoprecipitation of TARBP2 and its binding partners?

Immunoprecipitation (IP) of TARBP2 requires careful optimization, especially when studying its interactions with RNA or protein partners:

Lysis Buffer Selection:

• For protein-protein interactions: Use buffers containing 0.5% NP-40 or 1% Triton X-100

• For RNA-protein interactions: Consider crosslinking with formaldehyde before lysis

• Include protease and phosphatase inhibitors

Antibody Selection:

• Choose antibodies validated for IP applications

• Monoclonal antibodies often provide cleaner results with fewer non-specific interactions

Protocol Optimization:

• Pre-clear lysates with protein A/G beads to reduce background

• Use 2-5 μg of antibody per mg of total protein

• Incubate antibody-lysate mixture overnight at 4°C

• Thoroughly wash beads to remove non-specific binders

Co-IP Detection:

• When studying TARBP2 interactions with Dicer or Ago2, use stringent washing conditions

• For LGP2 interactions, gentler washing conditions may be required

• When investigating WTAP or METTL3 interactions, consider nuclear fractionation first

Research has shown that TARBP2 interacts specifically with LGP2 but not with related RIG-I-like receptors . Similarly, interactions between TARBP2 and the nucleoprotein TPR promote degradation of TARBP2-bound transcripts by the nuclear exosome .

How can I investigate the nuclear functions of TARBP2 in RNA splicing regulation?

Recent research has revealed that TARBP2 plays important roles in nuclear RNA processing, particularly in regulating splicing and RNA stability. To investigate these functions:

Nuclear-Cytoplasmic Fractionation:

• Use established protocols to separate nuclear and cytoplasmic fractions

• Verify fractionation quality using markers like GAPDH (cytoplasmic) and Lamin B (nuclear)

• Confirm TARBP2 presence in nuclear fractions via Western blot

RNA Immunoprecipitation (RIP):

• Use anti-TARBP2 antibodies to immunoprecipitate TARBP2-RNA complexes

• Extract and analyze bound RNAs via RT-PCR or RNA-seq

• Focus on intronic sequences, as TARBP2 shows pervasive binding to introns

Splicing Analysis:

• Use high-throughput transcriptomic profiling after TARBP2 knockdown

• Quantify intron retention using computational tools like MISO

• Calculate percent intron retention (PIR) changes in TARBP2-bound introns

Research has demonstrated that silencing TARBP2:

• Results in decreased retention of TARBP2-bound introns

• Increases expression of TARBP2 target transcripts in the nucleus

• Affects the stability of TARBP2-bound transcripts

m6A Methylation Analysis:

• Perform MeRIP-seq in control and TARBP2 knockdown cells

• Analyze changes in m6A methylation patterns of TARBP2 target RNAs

• Focus on TARBP2-bound introns and their flanking exons

Studies have shown that TARBP2-bound introns significantly overlap with introns that contain m6A marks, and TARBP2 knockdown leads to decreased m6A signal in these introns .

What is the role of TARBP2 in innate immune responses and how can it be studied using antibodies?

TARBP2 has been identified as an important factor in innate immune responses, particularly in virus-triggered interferon signaling. To study this function:

Protein-Protein Interaction Analysis:

• Use co-immunoprecipitation with anti-TARBP2 antibodies to pull down immune-related binding partners

• Perform Western blot analysis to detect interactions with specific proteins like LGP2

• Map interaction domains using truncated protein constructs

Research has shown that TARBP2 interacts specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5 . The interaction involves both dsRNA binding domains (dsRBD 1 and dsRBD 2) of TARBP2 .

Functional Analysis in Viral Infection:

• Perform TARBP2 knockdown experiments using siRNA

• Infect cells with different viruses (e.g., TMEV, EMCV, Sendai virus)

• Measure interferon responses via qRT-PCR and ELISA

• Use TARBP2 antibodies to monitor knockdown efficiency

Studies have demonstrated that silencing TARBP2 significantly reduces IFNβ mRNA induction upon Cardiovirus (TMEV, EMCV) infection but does not affect Sendai virus-triggered interferon response .

Quantitative Analysis of Cytokine Production:

This pattern suggests that TARBP2 is specifically involved in the LGP2/MDA5-mediated interferon response pathway rather than the RIG-I pathway .

Why might I observe multiple bands when performing Western blot for TARBP2?

Multiple bands in TARBP2 Western blots can result from several phenomena:

Alternative Splicing:

• TARBP2 has multiple transcript variants encoding different isoforms

• The two main isoforms (TRBP1 and TRBP2) differ slightly in size

• Verify which isoforms your antibody should detect based on the epitope location

Post-translational Modifications:

• TARBP2 can undergo phosphorylation which may cause mobility shifts

• Consider using phosphatase treatment of lysates to determine if bands are phosphorylated forms

Degradation Products:

• TARBP2 may undergo proteolytic processing during sample preparation

• Ensure fresh preparation of samples with adequate protease inhibitors

• Keep samples cold throughout processing

Antibody Cross-reactivity:

• Some antibodies may cross-react with related proteins like PACT/PRKRA

• Validate specificity using TARBP2 knockout or knockdown samples

• Compare results with multiple antibodies targeting different epitopes

Resolution Strategies:

• Optimize gel percentage to better separate closely migrating bands

• Run longer SDS-PAGE separations to distinguish closely-spaced bands

• Use gradient gels for improved resolution of multiple isoforms

• Compare band patterns with recombinant TARBP2 protein standards

How can I assess the specificity of a TARBP2 antibody in my experimental system?

To rigorously evaluate TARBP2 antibody specificity:

Genetic Validation:

• Generate TARBP2 knockdown or knockout samples

• Compare antibody signals between wild-type and TARBP2-depleted samples

• The specific signal should be significantly reduced or absent in knockdown/knockout samples

Peptide Competition:

• Pre-incubate the antibody with the immunizing peptide (if available)

• Perform Western blot or immunostaining in parallel with untreated antibody

• Specific signals should be blocked by peptide competition

Orthogonal Detection Methods:

• Compare results using multiple antibodies targeting different epitopes of TARBP2

• Use tagged TARBP2 constructs and detect with both anti-tag and anti-TARBP2 antibodies

• Concordant results increase confidence in antibody specificity

Positive Controls:

• Include recombinant TARBP2 protein in Western blots

• Use cell lines known to express high levels of TARBP2

• Compare molecular weight with the expected size (39 kDa for full-length TARBP2)

Immunoprecipitation-Mass Spectrometry:

• Perform IP with the TARBP2 antibody

• Analyze immunoprecipitated proteins by mass spectrometry

• TARBP2 should be among the top-identified proteins

• Known TARBP2 interactors (Dicer, Ago2, PACT) may also be detected

How can TARBP2 antibodies be utilized to study its role in cancer progression?

TARBP2 has been implicated in cancer development, particularly in lung cancer. To investigate its oncogenic roles:

Expression Analysis in Clinical Samples:

• Perform immunohistochemistry on tissue microarrays using validated TARBP2 antibodies

• Compare TARBP2 expression levels between normal and tumor tissues

• Correlate expression with clinical parameters and patient outcomes

Mechanistic Studies:

• Use TARBP2 antibodies for chromatin immunoprecipitation to identify upstream regulators

• Perform RNA immunoprecipitation to identify cancer-relevant TARBP2 RNA targets

• Conduct co-immunoprecipitation to map protein interaction networks in cancer cells

Research has identified ZNF143 as an upstream regulator of TARBP2 expression and shown that TARBP2-mediated destabilization of ABCA3 and FOXN3 impacts tumor growth in lung cancer models .

Functional Assays:

• Perform TARBP2 knockdown in cancer cell lines

• Use antibodies to verify knockdown efficiency

• Assess effects on cancer phenotypes (proliferation, migration, invasion)

• Analyze changes in RNA processing and stability of cancer-relevant transcripts

Studies demonstrate that increased activity of TARBP2 promotes lung cancer growth, and a network analytical approach has identified key factors that participate in its oncogenic role .

What approaches can be used to study the interaction between TARBP2 and the m6A methylation machinery?

Recent research has uncovered important connections between TARBP2 and RNA methylation. To investigate these interactions:

Protein Complex Analysis:

• Use TARBP2 antibodies for immunoprecipitation followed by Western blot

• Probe for m6A methyltransferase components (METTL3, METTL14, WTAP)

• Perform reciprocal IPs with antibodies against methyltransferase components

Research has shown that TARBP2 co-immunoprecipitates with METTL3, the enzymatic component of the methyltransferase complex .

Methylation Analysis:

• Perform MeRIP-seq in control and TARBP2 knockdown cells

• Quantify changes in m6A levels on TARBP2 target transcripts

• Focus on the overlap between TARBP2 binding sites and m6A modification sites

Studies have revealed that:

• More than 50% of TARBP2-bound introns contain methylation marks, compared to less than 10% of all expressed introns

• TARBP2 knockdown causes a significant decrease in m6A signal in TARBP2-bound introns

Functional Reporter Assays:

• Design minigene constructs containing TARBP2 binding sites

• Create variants with mutated binding sites

• Compare m6A levels and splicing patterns between wild-type and mutant constructs

• Assess the effects of TARBP2 or METTL3 knockdown on reporter expression

Experimental data has demonstrated that decreased intron retention upon reduced levels of TARBP2 and METTL3 is contingent on the presence of TARBP2 binding sites .

Mechanistic Model:
Current research suggests a model where TARBP2:

• Binds to intronic sequences in pre-mRNAs

• Recruits the m6A methyltransferase complex

• Facilitates m6A modification of target RNAs

• Inhibits efficient splicing of modified introns

• Promotes degradation of inefficiently spliced transcripts via interaction with nuclear exosome components