GTF2H1 Antibody

What is GTF2H1 and why is it important in molecular research?

GTF2H1 (General Transcription Factor IIH, Polypeptide 1, 62kDa) is a crucial component of the TFIIH complex involved in dual essential cellular processes: nucleotide excision repair (NER) of damaged DNA and RNA transcription by RNA polymerase II. In NER, TFIIH acts by opening DNA around lesions to facilitate excision of damaged oligonucleotides and their replacement with new DNA fragments. In transcription, GTF2H1 plays an essential role in initiation, where TFIIH is required for promoter opening and escape after pre-initiation complex (PIC) formation. The phosphorylation of the C-terminal domain of RNA polymerase II by the kinase module CAK controls transcription initiation .

This protein participates in a variety of important protein interactions. For example, retinoblastoma protein (Rb) competes with TATA-binding protein (TBP) and GTF2H1 for binding to E2F, thereby repressing E2F-mediated transactivation. Additionally, herpes simplex virus VP16 and human p53 directly interact with GTF2H1 .

What are the common types of GTF2H1 antibodies available for research?

GTF2H1 antibodies are available in multiple formats optimized for different experimental applications:

Most antibodies are unconjugated and purified using Protein A or antigen affinity chromatography. The immunogens used include KLH-conjugated synthetic peptides derived from human GTF2H1 and recombinant fragment proteins .

What is the difference between polyclonal and monoclonal GTF2H1 antibodies in experimental applications?

The choice between polyclonal and monoclonal GTF2H1 antibodies depends on experimental goals:

Polyclonal GTF2H1 antibodies:

• Recognize multiple epitopes on the GTF2H1 protein

• Provide higher sensitivity due to binding to multiple sites

• Useful for detection of denatured proteins in Western blotting

• Better for proteins with lower expression levels

• Show broader species cross-reactivity (e.g., recognize GTF2H1 in human, mouse, rat, and sometimes other species)

Monoclonal GTF2H1 antibodies:

• Recognize a single epitope (e.g., clone 1F12-1B5 targets AA 1-548)

• Offer higher specificity and lower background

• Provide more consistent results between batches

• Particularly useful for co-immunoprecipitation experiments where specificity is critical

• Typically have narrower species reactivity

For initial characterization or when working with low abundance samples, polyclonal antibodies may be preferable. For highly specific applications or when background is problematic, monoclonal antibodies are often the better choice.

How should GTF2H1 antibodies be validated for experimental use?

Proper validation of GTF2H1 antibodies is critical for generating reliable research data. A comprehensive validation approach includes:

• Western blot validation:

  • Verify band size matches the expected molecular weight (~62 kDa for full-length GTF2H1)

  • Use positive control cell lines (A549, HepG2, HL-60 cells show consistent expression)

  • Include negative controls (knockdown or knockout samples)

  • Test specificity with blocking peptides when available

• Cross-reactivity testing:

  • Determine reactivity across species if working with non-human models

  • Some antibodies show predicted reactivity with mouse, rat, dog, and other species

• Application-specific validation:

  • For immunohistochemistry: test on known positive tissues (testis tissue shows high expression)

  • For immunofluorescence: confirm subcellular localization pattern

  • For immunoprecipitation: verify pull-down efficiency with Western blot

A well-validated antibody should show consistent results across multiple experiments and match known biological characteristics of GTF2H1.

What are the optimal protocols for using GTF2H1 antibodies in Western blotting?

For optimal Western blot results with GTF2H1 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is important

• Gel separation and transfer:

  • 8-10% SDS-PAGE gels work well for the 62 kDa GTF2H1 protein

  • PVDF membranes generally provide better results than nitrocellulose

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Typical dilutions range from 1:500-1:2000 for primary antibody

  • Incubate primary antibody overnight at 4°C

  • Use species-appropriate HRP-conjugated secondary antibodies

• Detection considerations:

  • Enhanced chemiluminescence (ECL) detection works well

  • For weaker signals, consider using more sensitive ECL substrates

• Controls:

  • Include positive control lysates (A549, HepG2 cells)

  • Consider including recombinant GTF2H1 as a standard

The observed molecular weight is typically 62 kDa, matching the calculated molecular weight of GTF2H1 .

What are the best practices for immunohistochemistry with GTF2H1 antibodies?

For optimal immunohistochemistry (IHC) results with GTF2H1 antibodies:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections work well

  • Fresh-frozen sections can also be used

• Antigen retrieval:

  • Heat-induced epitope retrieval using TE buffer pH 9.0 is recommended

  • Alternative: citrate buffer pH 6.0

• Blocking and antibody incubation:

  • Block with normal serum corresponding to secondary antibody species

  • Use GTF2H1 antibody at dilutions of 1:100-1:500 for IHC

  • Incubate primary antibody overnight at 4°C

• Detection:

  • Use appropriate detection systems (HRP/DAB, AP/Fast Red)

  • Include DAPI or hematoxylin counterstain

• Controls:

  • Positive control tissues: testis tissue shows high expression

  • Negative controls: primary antibody omission or isotype control

For mouse tissues, consider using mouse-on-mouse detection systems to minimize background when using mouse monoclonal antibodies.

How can GTF2H1 antibodies be used to study DNA damage repair pathways?

GTF2H1 antibodies are valuable tools for investigating nucleotide excision repair (NER) pathways, as demonstrated in published research:

• Chromatin immunoprecipitation (ChIP) assays:

  • GTF2H1 antibodies can be used to study recruitment of TFIIH to sites of DNA damage

  • IP-grade antibodies are essential for these applications

• Immunofluorescence to track DNA repair kinetics:

  • Using GTF2H1 antibodies to visualize recruitment to UV-induced damage sites

  • Can be combined with other repair factor antibodies to study recruitment timing

• Analyzing NER in SWI/SNF-deficient cells:

  • Research has shown that SWI/SNF chromatin remodelers BRM and BRG1 promote GTF2H1 expression, impacting NER efficiency

  • GTF2H1 antibodies can be used to quantify protein levels in different genetic backgrounds

  • In BRM/BRG1-depleted cells, impaired TFIIH function was rescued by ectopic expression of GTF2H1

Research has demonstrated that DNA damage sensitivity of SWI/SNF-deficient cells depends on GTF2H1 levels, suggesting GTF2H1 as a potential predictive marker for platinum drug sensitivity in SWI/SNF-deficient cancer cells .

How can GTF2H1 antibodies be used to study virus-host interactions?

GTF2H1 antibodies can provide insights into virus-host interactions, as demonstrated in dengue virus research:

• Transcriptional response analysis:

  • Studies show that antibody-dependent dengue virus entry alters host responses that support the viral life cycle

  • GTF2H1 antibodies can help monitor changes in transcription factor complexes during infection

• Tracking TFIIH complex stability:

  • Using GTF2H1 antibodies to monitor TFIIH complex integrity during viral infection

  • Combined with antibodies against other TFIIH components to assess complex assembly

• Investigating viral manipulation of transcription:

  • Research indicates antibody-dependent virus entry can specifically induce genes associated with RNA splicing, including GTF2H1

  • This induction appears to enhance viral replication

When studying virus-host interactions, coupling GTF2H1 antibody techniques with transcriptomic analyses can provide more comprehensive understanding of how viruses manipulate host transcription machinery.

What role does GTF2H1 play in neurological development and disorders?

GTF2H1 and related transcription factors have been implicated in neurological development:

• Developmental studies:

  • Research on related transcription factors like Gtf2i and Gtf2ird1 shows they play important roles in developing brain

  • GTF2H1 antibodies can help characterize expression patterns in neural tissues

• Neurodevelopmental disorder models:

  • GTF2H1 is part of the TFIIH complex essential for transcription and DNA repair

  • Mutations affecting TFIIH components have been linked to neurodevelopmental disorders

  • Antibodies can help characterize expression in disease models

• DNA binding properties:

  • GTF2IRD1 binding targets are enriched for transcriptional and chromatin regulators

  • These targets overlap with CTCF binding and topological associating domain boundaries

  • Both share targets enriched for reactive oxygen species-responsive genes and synaptic proteins

Investigations using GTF2H1 antibodies in neural tissues require careful optimization of fixation and permeabilization protocols to maintain tissue architecture while enabling antibody penetration.

What are common issues with GTF2H1 antibodies in Western blotting and how can they be resolved?

When working with GTF2H1 antibodies in Western blotting, several common issues may arise:

• No signal or weak signal:

  • Increase antibody concentration (try 1:200-1:500 for weak signals)

  • Increase protein loading (20-40 μg total protein)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection substrates

  • Verify GTF2H1 expression in your specific sample type

• Multiple bands or non-specific binding:

  • Use more stringent washing (increase TBST washes to 4-5 times, 5-10 minutes each)

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Reduce primary antibody concentration

  • Verify antibody specificity with appropriate controls

• Unexpected molecular weight:

  • GTF2H1 should appear at approximately 62 kDa

  • Higher molecular weight bands may indicate post-translational modifications

  • Lower bands may indicate degradation or specific isoforms

  • Some antibodies recognize truncated forms of the protein

• High background:

  • Use fresher blocking reagents

  • Increase washing stringency

  • Reduce antibody concentration

  • Use higher quality secondary antibodies

For optimal results, store antibodies according to manufacturer recommendations (typically -20°C) and avoid repeated freeze-thaw cycles by preparing small aliquots.

How can immunohistochemistry with GTF2H1 antibodies be optimized for difficult tissue samples?

When working with challenging tissue samples for GTF2H1 immunohistochemistry:

• Fixation-resistant tissues:

  • Extended antigen retrieval (20-30 minutes)

  • Try alternative retrieval methods (pressure cooker, enzyme-based retrieval)

  • Consider dual retrieval approaches (heat followed by enzymatic)

• High background in specific tissues:

  • For liver, kidney, or brain tissues that often show high background:

    • Use longer blocking steps (2 hours)

    • Include additional blocking agents (avidin/biotin block if using biotinylated secondaries)

    • Consider tyramide signal amplification for specific signal enhancement

• Weak staining:

  • Titrate antibody concentration (try 1:50-1:200 range)

  • Extend primary antibody incubation to 48 hours at 4°C

  • Use polymer-based detection systems for signal enhancement

  • Consider paraffin section thickness (4-5 μm optimal)

• Tissue-specific considerations:

  • Brain tissues: additional permeabilization may be required

  • Testis tissue: shows high expression and works well as positive control

  • Paraffin vs. frozen sections: both work but may require different protocols

For challenging samples, performing a titration series with both antigen retrieval conditions and antibody concentrations can help identify optimal conditions.

What are the critical factors to consider when interpreting contradictory results from different GTF2H1 antibodies?

When faced with contradictory results from different GTF2H1 antibodies:

• Epitope differences:

  • Antibodies targeting different regions of GTF2H1 may give different results

  • N-terminal antibodies vs. central domain antibodies may detect different forms

  • Check if the epitopes are accessible in your experimental conditions

• Isoform specificity:

  • Confirm which isoforms your antibodies detect

  • Different antibodies may recognize different splice variants

• Post-translational modifications:

  • Research suggests GTF2H1 undergoes phosphorylation during mitosis

  • Modifications might mask epitopes for certain antibodies

  • For phosphorylation studies, compare results with phospho-specific antibodies

• Complex formation:

  • GTF2H1 exists in the TFIIH complex

  • Some epitopes may be masked when in complex

  • Native vs. denaturing conditions can give different results

• Cross-reactivity:

  • Validate species cross-reactivity if using different antibodies across model systems

  • Some antibodies have broader reactivity than others

When publishing contradictory results, include detailed methods sections specifying exact antibody catalog numbers, dilutions, and incubation conditions to enable proper replication.

How should experiments be designed to study GTF2H1 regulation by SWI/SNF chromatin remodelers?

Based on research showing GTF2H1 regulation by SWI/SNF chromatin remodelers BRM and BRG1, optimal experimental design should include:

• Gene expression analysis:

  • RT-qPCR to measure GTF2H1 mRNA levels following BRM/BRG1 knockdown

  • ChIP-qPCR using BRM/BRG1 antibodies to verify direct binding to GTF2H1 promoter

  • Analysis of other TFIIH subunits to assess specificity of regulation

• Protein level and stability assessment:

  • Western blotting with GTF2H1 antibodies following BRM/BRG1 depletion

  • Pulse-chase experiments to determine if protein stability is affected

  • Co-immunoprecipitation to assess TFIIH complex integrity

• Functional rescue experiments:

  • Complementation with ectopic GTF2H1 to rescue phenotypes

  • Testing nucleotide excision repair capacity

  • Analyzing transcriptional activity using reporter assays

• Cell type considerations:

  • Include multiple cell types, as some show adaptive mechanisms

  • Test both transient and stable knockdown models

  • Include cancer cell lines with SWI/SNF mutations (e.g., A549, H1299)

Research has shown that DNA damage sensitivity in SWI/SNF-deficient cells correlates with GTF2H1 levels, suggesting GTF2H1 could potentially be used as a marker for platinum drug sensitivity in SWI/SNF-deficient cancers .

What experimental controls are essential when studying GTF2H1 function in DNA repair pathways?

When investigating GTF2H1's role in DNA repair, include these essential controls:

• Positive controls:

  • Known NER-deficient cells (XPA-/-, XPC-/-)

  • UV-sensitive cell lines

  • Cells treated with known TFIIH inhibitors

• Negative controls:

  • Complemented cells (GTF2H1-deficient cells with restored expression)

  • Isogenic wild-type cells

  • Cells with mutations in non-NER pathways

• Technical controls:

  • Antibody specificity controls (blocking peptides, isotype controls)

  • siRNA off-target controls (multiple siRNAs, rescue experiments)

  • DNA damage verification (CPD antibodies for UV damage)

• Functional readouts:

  • Direct repair assays (Unscheduled DNA synthesis, Host cell reactivation)

  • Survival assays with DNA damaging agents

  • TFIIH complex integrity assessment

Research has demonstrated that XPD recruitment to local UV damage was impaired in BRM and BRG1 depleted cells, but could be rescued by ectopic expression of GTF2H1, confirming specificity of the observed repair defects .

How can GTF2H1 antibodies be used to differentiate between its roles in transcription versus DNA repair?

Distinguishing GTF2H1's dual functions requires carefully designed experiments:

• Chromatin association studies:

  • ChIP-seq to identify genome-wide binding sites during transcription vs. repair

  • Compare GTF2H1 binding before and after DNA damage induction

  • Co-occupancy analysis with transcription vs. repair factors

• Mutational analysis:

  • Distinguish domain-specific functions using domain-specific antibodies

  • Correlate antibody binding with specific functional outcomes

• Temporal dynamics:

  • Time-course experiments following DNA damage

  • Synchronized cell populations to separate cell-cycle effects

• Co-localization studies:

  • Combine with markers specific to transcription (RNA Pol II phospho-CTD)

  • Compare with repair-specific markers (γH2AX, XPC)

  • Triple staining to identify transition points between functions

• Functional separation:

  • Use α-amanitin to inhibit transcription while studying repair function

  • Use specifically timed UV irradiation to study repair without affecting global transcription

Research has shown that GTF2H1 depletion impacts both transcription levels and DNA repair capacity, with ectopic expression of GTF2H1 rescuing XPD recruitment to local UV damage sites .

How should researchers interpret GTF2H1 expression changes in disease models?

When analyzing GTF2H1 expression changes in disease contexts:

• Cancer studies:

  • Decreased GTF2H1 may indicate compromised DNA repair capacity

  • SWI/SNF mutations often impact GTF2H1 expression

  • Chronic vs. acute changes may have different implications due to adaptive mechanisms

• Neurodegenerative diseases:

  • Changes may reflect altered transcriptional regulation

  • Consider impact on both RNA Pol II transcription and DNA repair capacity

  • Correlate with markers of DNA damage accumulation

• Viral infections:

  • Altered GTF2H1 expression during viral infection may indicate viral manipulation of host transcription machinery

  • Antibody-dependent viral entry can specifically induce genes involved in RNA splicing, including GTF2H1

• Developmental disorders:

  • Consider tissue-specific expression patterns

  • Related transcription factors (Gtf2i and Gtf2ird1) play roles in neurological development

• Quantification approaches:

  • Use both mRNA (RT-qPCR) and protein (Western blot) quantification

  • Consider complex formation and stability

  • Normalize to appropriate housekeeping genes/proteins

When publishing GTF2H1 expression data from disease models, include detailed information about antibody validation, quantification methods, and statistical analysis to ensure reproducibility.

What emerging technologies are enhancing GTF2H1 antibody-based research?

Several innovative technologies are advancing GTF2H1 antibody applications:

• Proximity labeling approaches:

  • BioID or APEX2 fusions with GTF2H1 to identify context-specific interaction partners

  • Allows identification of transient interactions during transcription vs. repair

• Live-cell imaging techniques:

  • Antibody fragments (nanobodies) for live-cell visualization

  • CRISPR-based tagging for endogenous protein tracking

  • Super-resolution microscopy to visualize TFIIH complex assembly

• Single-cell applications:

  • Mass cytometry (CyTOF) with GTF2H1 antibodies for single-cell protein analysis

  • Integration with single-cell transcriptomics

  • Spatial transcriptomics combined with GTF2H1 immunostaining

• Automated high-content screening:

  • High-throughput imaging of GTF2H1 recruitment to damage sites

  • Screening for compounds that modulate TFIIH function

  • Machine learning algorithms to classify phenotypes

• CRISPR-based genetic screens:

  • Combining CRISPR screens with GTF2H1 antibody-based readouts

  • Identifying novel regulators of TFIIH complex assembly and function

These emerging technologies offer opportunities to study GTF2H1 with unprecedented spatial and temporal resolution in both normal and disease states.

What are the current limitations in GTF2H1 antibody research and how might they be addressed?

Current limitations in GTF2H1 antibody research include:

• Specificity challenges:

  • Cross-reactivity with related proteins in some species

  • Solution: Develop and validate highly specific monoclonal antibodies

  • CRISPR knockout validation to confirm specificity

• Post-translational modification detection:

  • Limited availability of modification-specific antibodies

  • Solution: Develop antibodies against known GTF2H1 modifications

  • Combine with mass spectrometry for comprehensive PTM mapping

• Complex assembly detection:

  • Difficulty distinguishing free vs. TFIIH-complexed GTF2H1

  • Solution: Develop conformation-specific antibodies

  • Proximity ligation assays to visualize complex formation

• Quantification standardization:

  • Variability in antibody performance between lots

  • Solution: Develop recombinant protein standards

  • Establish quantitative benchmarks across laboratories

• Tissue penetration in thick sections:

  • Limited antibody penetration in brain and other dense tissues

  • Solution: Develop smaller antibody fragments

  • Tissue clearing techniques compatible with immunostaining

Future directions should include development of highly specific recombinant antibodies with standardized validation protocols to enhance reproducibility across laboratories.