GTF2A2 Antibody

What is GTF2A2 and what is its biological function?

GTF2A2 is the gamma subunit (12 kDa) of the general transcription factor IIA complex. Transcription mediated by RNA polymerase II depends on multiple transcription factors forming the pre-initiation complex, with TFIIA playing a crucial role in this assembly. Specifically, TFIIA increases the affinity of TATA-binding protein (TBP) for the DNA union region . GTF2A2 functions as one of the subunits of TFIIA, which in complex with TBP mediates transcriptional activity .

In human cells, the TFIIA complex consists of three subunits: a 35-kD alpha subunit and a 19-kD beta subunit (both encoded by GTF2A1), and a 12-kD gamma subunit encoded by GTF2A2 . This transcription factor is essential for accurate transcription initiation on TATA-containing class II genes, making it fundamental to normal cellular gene expression .

What are the primary applications for GTF2A2 antibodies?

GTF2A2 antibodies are utilized in multiple experimental applications including:

Researchers should note that optimal dilutions and conditions may vary between antibodies from different manufacturers and should be determined empirically for each specific experimental setup .

What is the subcellular localization of GTF2A2?

Immunocytochemistry/immunofluorescence studies have demonstrated that GTF2A2 is predominantly localized to the nucleoplasm, consistent with its role in transcriptional regulation . This has been specifically observed in human cell lines such as U-2 OS, where staining with GTF2A2 antibodies clearly shows nuclear localization . This subcellular distribution is expected given GTF2A2's function in the pre-initiation complex formation for RNA polymerase II-mediated transcription.

Which species do GTF2A2 antibodies typically detect?

Most commercially available GTF2A2 antibodies demonstrate reactivity with:

The high sequence conservation of GTF2A2 across mammalian species enables many antibodies to cross-react with multiple species. For example, Proteintech's antibody (10540-1-AP) has been experimentally validated to react with human, mouse, and rat samples , while Bio-Techne's antibody (NBP2-55408) is validated for human and predicted to work with mouse and rat due to the 99% sequence homology .

How should Western blot protocols be optimized for GTF2A2 detection?

When performing Western blot analysis for GTF2A2, researchers should consider the following optimization strategies:

• Sample Preparation: GTF2A2 is a nuclear protein, so efficient nuclear extraction techniques are critical. Standard RIPA or nuclear extraction buffers with protease inhibitors are recommended.

• Gel Selection: Use higher percentage gels (12-15%) as GTF2A2 is a small protein with an observed molecular weight of approximately 12 kDa .

• Transfer Conditions: Employ semi-dry or wet transfer with careful optimization for small proteins. Extended transfer times at lower voltages may improve transfer efficiency.

• Blocking: Use 5% non-fat dry milk or BSA in TBST. Some antibodies may perform better with specific blocking reagents.

• Antibody Dilution: Start with recommended dilutions:

  • Proteintech 10540-1-AP: 1:200-1:1000

  • Elabscience E-AB-62777: 1:500-1:2000

  • Bio-Techne NBP2-55408: 0.04-0.4 μg/ml

• Positive Controls: Include validated positive controls such as HeLa cells, NCI-H460 cells, RT-4 cells or U-251 MG cells, which have been documented to express detectable levels of GTF2A2 .

Western blot analysis typically reveals a distinct band at approximately 12 kDa, which corresponds to the calculated molecular weight of GTF2A2 .

What are recommended fixation and permeabilization methods for immunohistochemistry/immunocytochemistry?

For optimal GTF2A2 detection in tissue and cell preparations:

• Tissue Fixation for IHC:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • Antigen retrieval is critical: use TE buffer pH 9.0 as preferred method, with citrate buffer pH 6.0 as an alternative

• Cell Fixation for ICC/IF:

  • Paraformaldehyde (PFA) fixation (typically 4%) for 10-15 minutes

  • Permeabilization with Triton X-100 is recommended

• Antibody Incubation:

  • For IHC: Recommended dilutions range from 1:500-1:2000

  • For ICC/IF: Typically 0.25-2 μg/ml

• Detection Systems:

  • Both chromogenic (HRP/DAB) and fluorescent secondary detection systems are compatible

  • For fluorescent detection, select fluorophores that avoid spectral overlap with nuclear counterstains for clear visualization of the nuclear signal

When performing these techniques, it's important to include both positive and negative controls to validate staining specificity .

How should GTF2A2 antibodies be stored to maintain optimal activity?

Proper storage of GTF2A2 antibodies is crucial for maintaining their reactivity and specificity:

• Storage Temperature:

  • Long-term storage: -20°C is recommended for most antibodies

  • Some manufacturers suggest -20°C even for opened vials

  • Avoid repeated freeze-thaw cycles by aliquoting antibodies upon receipt

• Buffer Conditions:

  • Most GTF2A2 antibodies are supplied in PBS with glycerol (typically 50%) and a preservative such as 0.02% sodium azide

  • The glycerol prevents freezing at -20°C and helps maintain antibody stability

• Stability:

  • Most GTF2A2 antibodies remain stable for at least one year after shipment when stored properly

  • For antibodies without BSA, some manufacturers include glycerol at higher concentrations (e.g., 40%) for stabilization

• Working Dilutions:

  • Prepare fresh working dilutions on the day of use

  • Avoid storing diluted antibody for extended periods

Following these storage recommendations will help ensure consistent and reliable results across experiments .

How can GTF2A2 antibodies be utilized in chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation with GTF2A2 antibodies can provide valuable insights into transcription factor binding and regulation. For optimal ChIP experiments:

• Experimental Design:

  • GTF2A2 antibodies can be used to study its association with specific promoter regions in vivo

  • Consider dual ChIP approaches to examine co-occupancy with other transcription factors like TBP

• Protocol Optimization:

  • Crosslinking: Standard 1% formaldehyde for 10 minutes at room temperature

  • Chromatin shearing: Optimization for fragments of 200-500 bp is critical

  • Antibody amounts: Typical range is 2-5 μg per ChIP reaction, but should be empirically determined

  • Include appropriate controls: IgG negative control and a positive control antibody targeting a known abundant transcription factor

• Applications:

  • Study association patterns at RNA polymerase II-transcribed genes

  • Investigate temporal dynamics of pre-initiation complex assembly

  • Examine GTF2A2 recruitment during different cellular conditions or treatments

Previous research has successfully employed TFIIA antibodies in ChIP assays to study the association of human autoantigen La with pol III-transcribed genes in vivo , demonstrating the feasibility of this approach for GTF2A2-related investigations.

What are the best methods for validating GTF2A2 antibody specificity?

Ensuring antibody specificity is critical for accurate data interpretation. Comprehensive validation should include:

• Western Blot Analysis:

  • Verify correct molecular weight (12 kDa for GTF2A2)

  • Include positive controls such as HeLa, NCI-H460, RT-4, or U-251 MG cell lines

  • Consider using tissues with known expression (e.g., mouse testis)

• Knockdown/Knockout Validation:

  • siRNA or CRISPR-mediated knockdown/knockout of GTF2A2 should result in diminished or absent signal

  • This negative control is the gold standard for antibody validation

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Should result in signal reduction/elimination in subsequent applications

• Cross-Reactivity Assessment:

  • Test antibody in species beyond those recommended to determine cross-reactivity limits

  • Evaluate potential cross-reactivity with other TFIIA components or related transcription factors

• Multiple Antibody Comparison:

  • Use multiple antibodies targeting different epitopes of GTF2A2

  • Concordant results with different antibodies increase confidence in specificity

• Recombinant Protein Controls:

  • Test antibody against purified recombinant GTF2A2 protein

  • Both positive detection of target and lack of reactivity with other recombinant proteins should be demonstrated

These validation approaches should be documented and considered when interpreting experimental results to ensure the reliability and reproducibility of findings with GTF2A2 antibodies .

How does GTF2A2 interact with other factors in the transcription pre-initiation complex?

GTF2A2 functions within the larger context of transcriptional regulation through multiple protein-protein interactions:

• Interaction with TBP:

  • TFIIA, including the GTF2A2 subunit, increases the affinity of TATA-binding protein (TBP) for the DNA binding region

  • This stabilization is crucial for proper transcription initiation

• Pre-Initiation Complex Formation:

  • GTF2A2 is involved in the ordered assembly with RNA polymerase II and other general initiation factors including TFIIB, TFIID, TFIIE, TFIIF, TFIIG/TFIIJ, and TFIIH

  • This assembly occurs at TATA-containing class II gene promoters

• Structural Considerations:

  • In human cells, TFIIA consists of three subunits:

    • 35-kD alpha subunit (N-terminal portion of GTF2A1)

    • 19-kD beta subunit (C-terminal portion of GTF2A1)

    • 12-kD gamma subunit (GTF2A2)

  • The complete complex is essential for transcriptional activity

• Regulatory Interactions:

  • TFIIA may interact with TBP-associated factors (TAFs) in TFIID, enabling more complex regulatory control

  • These interactions contribute to transcriptional regulation in response to various cellular signals

Understanding these interactions is important when designing experiments to study GTF2A2 function, particularly when considering co-immunoprecipitation studies or when interpreting results from chromatin immunoprecipitation experiments .

What approaches are available for studying post-translational modifications of GTF2A2?

Post-translational modifications (PTMs) can significantly impact GTF2A2 function. Several approaches can be employed to study these modifications:

• Modification-Specific Antibodies:

  • While not specifically mentioned in the search results, researchers should consider antibodies targeting specific PTMs (phosphorylation, acetylation, etc.)

  • Western blotting with these antibodies can identify the presence and abundance of modified GTF2A2

• Mass Spectrometry Analysis:

  • Immunoprecipitate GTF2A2 using validated antibodies

  • Subject the precipitated protein to tryptic digestion and LC-MS/MS analysis

  • This approach can identify multiple modifications simultaneously and provide site-specific information

• Electrophoretic Mobility Techniques:

  • Phos-tag gels or 2D gel electrophoresis to separate modified forms

  • Can be followed by western blotting with GTF2A2 antibodies

• Functional Assays:

  • Site-directed mutagenesis of potential modification sites

  • Comparison of wild-type and mutant GTF2A2 in transcriptional activity assays

• Temporal Dynamics:

  • Studying changes in modifications under different cellular conditions or treatments

  • Utilizing synchronization protocols to examine cell cycle-dependent modifications

These approaches can provide important insights into how GTF2A2 function is regulated through post-translational modifications, potentially revealing new mechanisms in transcriptional control.

How can non-specific binding be reduced in GTF2A2 antibody applications?

Non-specific binding can compromise experimental results. Several strategies can minimize this issue:

• Blocking Optimization:

  • Test different blocking agents (BSA, non-fat dry milk, commercial blockers)

  • Extend blocking times (1-2 hours at room temperature or overnight at 4°C)

  • Consider adding 0.1-0.3% Tween-20 or Triton X-100 to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Titrate antibody concentrations to find optimal signal-to-noise ratio

  • Follow manufacturer's recommended ranges as starting points:

    • Western blot: 1:200-1:2000

    • IHC: 1:500-1:2000

    • ICC/IF: 0.25-2 μg/ml

• Buffer Modifications:

  • Add 0.1-0.5M NaCl to reduce ionic interactions

  • Consider adding 0.1-1% BSA to antibody dilution buffers

  • For some applications, adding 5-10% normal serum from the secondary antibody host species may help

• Sample Preparation:

  • Ensure complete lysis and denaturation for western blot

  • Optimize fixation and antigen retrieval for IHC/ICC

  • For IHC, consider alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Controls:

  • Always include negative controls (secondary antibody only, isotype control)

  • Use competing peptide controls when available

Implementation of these strategies should be systematically recorded and evaluated to establish optimal conditions for specific experimental setups.

What are typical challenges when working with GTF2A2 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with GTF2A2 antibodies:

• Detection of Low Abundance Protein:

  • Challenge: GTF2A2 may be expressed at low levels in some cell types

  • Solution: Increase protein loading for western blots, use sensitive detection systems (ECL-Plus, fluorescent secondary antibodies), consider enrichment by nuclear fractionation

• Cross-Reactivity with Related Proteins:

  • Challenge: Antibodies may detect other TFIIA subunits or related transcription factors

  • Solution: Validate with knockdown controls, use multiple antibodies targeting different epitopes, perform peptide competition assays

• Variability Between Tissue/Cell Types:

  • Challenge: Expression and detection may vary significantly between samples

  • Solution: Optimize protocols for each specific sample type, use positive controls (e.g., HeLa cells, mouse testis tissue)

• Epitope Masking:

  • Challenge: Protein-protein interactions may mask epitopes, particularly in ChIP applications

  • Solution: Test different antibodies targeting different epitopes, optimize crosslinking conditions, consider native ChIP approaches

• Antibody Lot-to-Lot Variability:

  • Challenge: Performance may vary between antibody batches

  • Solution: Validate each new lot against previous lots, maintain detailed records of antibody performance

Addressing these challenges requires systematic optimization and thorough documentation of experimental conditions for reproducible results.

How can researchers quantitatively analyze GTF2A2 levels across different experimental conditions?

Accurate quantification of GTF2A2 is essential for comparative studies. Several approaches can be employed:

• Western Blot Quantification:

  • Use digital imaging systems with linear dynamic range

  • Include housekeeping protein controls (tubulin, actin) or total protein stains (Ponceau S, SYPRO Ruby)

  • Apply normalization algorithms to account for loading differences

  • Consider using standard curves with recombinant GTF2A2 protein for absolute quantification

• Quantitative Immunofluorescence:

  • Use consistent acquisition parameters (exposure time, gain)

  • Include calibration standards in each experiment

  • Employ nuclear counterstains for normalization

  • Utilize automated image analysis software for unbiased quantification

• ChIP-qPCR Analysis:

  • Design primers targeting known GTF2A2 binding regions

  • Normalize to input DNA and IgG controls

  • Consider percent input method or fold enrichment calculations

  • Include positive and negative control regions

• Proteomics Approaches:

  • SILAC, TMT, or iTRAQ labeling for mass spectrometry-based quantification

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for targeted quantification

  • Include internal standard peptides for absolute quantification

• ELISA Development:

  • Sandwich ELISA using capture and detection antibodies targeting different GTF2A2 epitopes

  • Establish standard curves using recombinant GTF2A2 protein

  • Validate assay linearity, sensitivity, and specificity

These quantitative approaches enable researchers to accurately measure changes in GTF2A2 levels or binding patterns across different experimental conditions, tissues, or disease states.

How might GTF2A2 antibodies contribute to understanding disease mechanisms?

GTF2A2 antibodies can provide valuable insights into transcriptional dysregulation in various diseases:

• Cancer Research:

  • Investigate alterations in transcription factor recruitment and function

  • Examine GTF2A2 expression or localization changes in different cancer types

  • Study how oncogenic signaling pathways impact pre-initiation complex assembly

• Developmental Disorders:

  • Explore GTF2A2 function in developmental gene expression programs

  • Investigate potential mutations or dysregulation in congenital disorders

• Neurological Conditions:

  • Study GTF2A2 involvement in neuron-specific gene expression

  • Examine potential roles in neurodegenerative diseases

• Inflammatory Diseases:

  • Investigate GTF2A2 roles in inflammatory gene expression

  • Examine potential targeting by autoantibodies in autoimmune conditions

• Drug Development:

  • Screen compounds that modulate transcription factor interactions

  • Validate drug targets in the transcriptional machinery

Future research using GTF2A2 antibodies could help elucidate fundamental disease mechanisms and potentially identify novel therapeutic targets in the transcriptional machinery.

What emerging technologies might enhance GTF2A2 research?

Several cutting-edge approaches hold promise for advancing GTF2A2 research:

• Single-Cell Techniques:

  • Single-cell ChIP-seq to examine cell-to-cell variability in GTF2A2 binding

  • Single-cell proteomics to quantify GTF2A2 levels in rare cell populations

• Proximity Labeling:

  • BioID or APEX2 fusions to identify GTF2A2 protein interaction networks

  • TurboID for temporal mapping of dynamic interactions

• Live-Cell Imaging:

  • CRISPR-mediated tagging of endogenous GTF2A2 with fluorescent proteins

  • Real-time visualization of transcription factor dynamics

• Cryo-EM and Structural Studies:

  • High-resolution structural analysis of GTF2A2 within the pre-initiation complex

  • Structure-guided development of specific antibodies targeting functional domains

• Genome Engineering:

  • CRISPR interference/activation to modulate GTF2A2 expression

  • Precise mutation of GTF2A2 genomic loci to study variant effects

• Spatial Transcriptomics:

  • Combining GTF2A2 immunostaining with spatial RNA sequencing

  • Correlating transcription factor localization with gene expression patterns

These emerging technologies, when combined with high-quality GTF2A2 antibodies, may provide unprecedented insights into transcriptional regulation mechanisms and their dysregulation in disease states.