GTF2H1 Monoclonal Antibody

What is GTF2H1 and what cellular functions does it perform?

GTF2H1 (General Transcription Factor IIH Subunit 1) is a critical component of the general transcription and DNA repair factor IIH (TFIIH) core complex. It serves dual functions in the cellular environment: it participates in nucleotide excision repair (NER) of damaged DNA and, when complexed with the CDK-activating kinase (CAK), facilitates RNA transcription by RNA polymerase II. In NER, TFIIH opens DNA around lesions to allow excision of damaged oligonucleotides and their replacement with new DNA fragments. In transcription, GTF2H1 as part of TFIIH is essential for transcription initiation, specifically for promoter opening and promoter escape during the formation of the pre-initiation complex (PIC). Furthermore, phosphorylation of the C-terminal domain (CTD) of RNA polymerase II's largest subunit by the CAK module controls transcription initiation .

How does GTF2H1 interact with other components of the TFIIH complex?

GTF2H1 serves as a structural scaffold within the TFIIH complex, physically interacting with multiple components to maintain complex stability and function. It contacts TFIIE of the transcriptional pre-initiation complex, facilitating transcription initiation. In nucleotide excision repair pathways, GTF2H1 interacts directly with DNA damage recognition factors such as XPC-HR23B and the 3′-endonuclease XPG through a pleckstrin homology domain . These interactions are crucial for the assembly and stability of the TFIIH complex, which subsequently impacts both general transcription and DNA repair capacity. The bridge-like function of GTF2H1 allows it to coordinate the dual activities of TFIIH in maintaining genomic and transcriptional homeostasis .

What is the regulatory relationship between MITF and GTF2H1?

The Microphthalmia-associated transcription factor (MITF) directly controls GTF2H1 expression through transcriptional regulation. Research has demonstrated that MITF binds to three conserved E-box elements in the GTF2H1 promoter region, located at positions -34, -67, and -340 base pairs upstream of the transcriptional start site. Through ChIP assays, studies have confirmed MITF occupancy at these sites . When MITF is depleted through RNA interference or dominant-negative MITF mutants, GTF2H1 expression is substantially reduced at both mRNA and protein levels, particularly under serum starvation conditions. This regulatory relationship is further evidenced by the concordant expression of MITF and GTF2H1 transcripts in various melanoma cell lines. Additionally, stimulation with Stem Cell Factor (SCF), which activates MITF via c-KIT/MAP kinase signaling, leads to increased GTF2H1 expression, an effect that is abrogated when MITF is depleted .

What are the validated applications for GTF2H1 monoclonal antibodies in research?

GTF2H1 monoclonal antibodies have been validated for several research applications, primarily in protein detection and localization studies. These applications include Western blotting (WB) for protein expression analysis, immunohistochemistry on paraffin-embedded tissues (IHC-P) for in situ detection in tissue samples, and immunocytochemistry/immunofluorescence (ICC/IF) for cellular localization studies . These antibodies are particularly valuable for studying TFIIH complex assembly, DNA repair mechanisms, and transcriptional regulation. They can be employed to investigate GTF2H1 expression levels in different cell types or tissues, to assess changes in expression following experimental manipulations such as UV radiation exposure, and to study protein-protein interactions through co-immunoprecipitation experiments. The specificity of these antibodies for human GTF2H1 makes them particularly suitable for translational research involving human cell lines and tissue samples .

How can GTF2H1 antibodies be used to study nucleotide excision repair (NER) mechanisms?

GTF2H1 antibodies provide valuable tools for investigating nucleotide excision repair mechanisms through several experimental approaches. They can be used to quantitatively assess NER capacity in relation to GTF2H1 expression by combining antibody-based detection with unscheduled DNA synthesis (UDS) assays that utilize 5-ethylene-2′-deoxyuridine (EdU) incorporation. This approach can reveal how GTF2H1 levels correlate with repair efficiency . Additionally, GTF2H1 antibodies can be employed in immunoprecipitation studies to examine interactions with other NER components such as XPG and damage recognition factors like XPC-HR23B. For studying repair kinetics, researchers can use GTF2H1 antibodies alongside detection methods for cyclobutane pyrimidine dimers (CPDs) or cisplatin-induced DNA adducts to track repair progression over time following DNA damage . Combining these techniques with siRNA-mediated knockdown or overexpression of GTF2H1 can establish causal relationships between GTF2H1 levels and NER capacity, offering insights into how transcription factors like MITF influence DNA repair through GTF2H1 regulation.

How can researchers distinguish between GTF2H1's roles in transcription versus DNA repair using antibody-based approaches?

Distinguishing between GTF2H1's dual roles in transcription and DNA repair requires sophisticated experimental design using antibody-based approaches. One effective strategy involves chromatin immunoprecipitation (ChIP) with GTF2H1 antibodies followed by sequencing (ChIP-seq) under different cellular conditions. By comparing GTF2H1 binding patterns before and after UV irradiation, researchers can distinguish between transcription-associated binding (stable across conditions) and repair-specific recruitment (enriched at damage sites after UV exposure) . Another approach combines GTF2H1 immunoprecipitation with mass spectrometry to identify differential protein interactions under transcription-favoring versus repair-inducing conditions. Researchers can also utilize GTF2H1 antibodies in combination with transcription inhibitors like 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) to determine if GTF2H1's recruitment to DNA damage sites occurs independently of active transcription . Furthermore, time-course immunofluorescence studies following UV irradiation can reveal the temporal dynamics of GTF2H1 relocalization, helping differentiate between its constitutive role in transcription and its inducible function in DNA repair.

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

For optimal Western blotting with GTF2H1 monoclonal antibodies, researchers should implement a comprehensive protocol that addresses several critical parameters. Sample preparation should begin with efficient cell lysis using a buffer containing 1% NP-40 or Triton X-100, 150mM NaCl, 50mM Tris-HCl (pH 8.0), and protease inhibitors. Given that GTF2H1 is a nuclear protein, nuclear extraction protocols may yield cleaner results with less cytoplasmic contamination. For protein separation, 8-10% polyacrylamide gels are recommended to achieve optimal resolution of GTF2H1, which has a molecular weight of approximately 62 kDa . Transfer to nitrocellulose or PVDF membranes should be conducted at 100V for 60-90 minutes in transfer buffer containing 20% methanol. Blocking should employ 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Primary antibody incubation should be conducted at dilutions of 1:500 to 1:2000 in blocking buffer, optimally overnight at 4°C. Following thorough washing, an appropriate HRP-conjugated secondary antibody should be applied at 1:5000-1:10000 dilution for 1 hour at room temperature. Detection via enhanced chemiluminescence should produce a specific band at 62 kDa, with validation through positive controls like HeLa or melanoma cell lysates that express GTF2H1 at detectable levels .

How can researchers effectively use GTF2H1 antibodies for immunocytochemistry/immunofluorescence studies?

For effective immunocytochemistry/immunofluorescence studies using GTF2H1 antibodies, researchers should follow a methodically optimized protocol. Begin by growing cells on sterile coverslips to 70-80% confluence, followed by fixation with 4% paraformaldehyde for 15 minutes at room temperature. After PBS washes, permeabilize cells with 0.2% Triton X-100 in PBS for 10 minutes. Blocking should be performed with 5% normal serum (from the same species as the secondary antibody) containing 0.1% Triton X-100 for 1 hour. Incubate with primary GTF2H1 antibody at a 1:100 to 1:500 dilution in blocking buffer overnight at 4°C . After washing, apply fluorophore-conjugated secondary antibodies at 1:500 dilution for 1 hour at room temperature in darkness. For nuclear visualization, counterstain with DAPI (1 μg/ml) for 5 minutes. Mount coverslips using anti-fade mounting medium. For optimal results, include internal controls such as TFIIH subunits known to colocalize with GTF2H1. To visualize DNA repair complexes, consider treating cells with UV radiation (10-20 J/m²) prior to fixation, which should induce punctate nuclear patterns of GTF2H1 accumulation at damage sites . This can be combined with EdU incorporation assays to simultaneously assess repair synthesis and GTF2H1 localization.

What are the most effective approaches for validating GTF2H1 antibody specificity?

Validating GTF2H1 antibody specificity requires a multi-faceted approach combining several complementary techniques. First, researchers should perform Western blot analysis across multiple cell lines with known varying levels of GTF2H1 expression, confirming the presence of a single band at the expected molecular weight of 62 kDa . This should be compared with siRNA-mediated knockdown of GTF2H1, which should result in significantly reduced band intensity. For definitive validation, researchers can use CRISPR/Cas9-mediated knockout cell lines as negative controls. Immunoprecipitation followed by mass spectrometry should confirm that the antibody pulls down GTF2H1 and its known interaction partners from the TFIIH complex. For immunocytochemistry applications, researchers should verify the expected nuclear localization pattern and demonstrate colocalization with other TFIIH components. Additionally, peptide competition assays using the immunizing peptide (corresponding to amino acids 50-200 of human GTF2H1) should block antibody binding in all applications if the antibody is specific . Cross-reactivity testing across species should be conducted when working with non-human models, as sequence variations may affect epitope recognition. Finally, parallel testing with alternative antibodies targeting different epitopes of GTF2H1 should yield consistent results across various applications.

How should researchers interpret contradictory results between GTF2H1 expression and nucleotide excision repair capacity?

When interpreting contradictory results between GTF2H1 expression and nucleotide excision repair (NER) capacity, researchers should conduct a systematic analysis considering several key factors. First, examine the cell-type specific context, as the relationship between GTF2H1 levels and repair capacity may vary across different cellular backgrounds due to differential expression of other TFIIH components or regulatory factors like MITF . Assess the integrity of the entire TFIIH complex through co-immunoprecipitation studies, as GTF2H1 expression alone may not reflect functional TFIIH assembly. Consider post-translational modifications of GTF2H1, which may affect its activity without altering total protein levels. Evaluate the experimental timing carefully, as transient versus stable alterations in GTF2H1 expression may have different consequences for repair capacity. Determine if the apparent contradiction relates to global NER or specifically to transcription-coupled repair (TCR) versus global genomic repair (GGR) pathways, as GTF2H1 may differentially impact these sub-pathways . Analyze the specific DNA damage type and dose used to assess repair, as the relationship between GTF2H1 and repair efficiency may be damage-specific. Finally, consider compensatory mechanisms that might be activated upon GTF2H1 alteration, such as upregulation of alternative repair pathways or other TFIIH components, particularly in long-term studies where cells have had time to adapt to altered GTF2H1 levels .

What controls should be included when assessing GTF2H1 antibody staining patterns in cancer versus normal tissues?

When assessing GTF2H1 antibody staining patterns in cancer versus normal tissues, researchers should implement a comprehensive set of controls to ensure reliable interpretation. Include isotype controls matched to the GTF2H1 antibody's species and immunoglobulin class to identify non-specific binding. Incorporate tissue microarrays (TMAs) containing both malignant and corresponding normal tissues to allow direct comparison under identical staining conditions . Use siRNA-treated or CRISPR-edited cell lines with confirmed GTF2H1 knockdown/knockout embedded in paraffin blocks as negative controls. Include positive control tissues with known GTF2H1 expression, such as proliferating epithelial tissues or melanoma samples with confirmed MITF expression . Perform antibody titration experiments to determine optimal concentrations that minimize background while maintaining specific signal. Implement antigen retrieval controls to ensure that observed differences are not artifacts of differential epitope accessibility. Include comparative staining with antibodies against other TFIIH components to determine whether changes are GTF2H1-specific or reflect alterations in the entire complex. Validate key findings using orthogonal methods such as in situ hybridization for GTF2H1 mRNA or Western blotting of protein extracts from the same tissue types . Lastly, conduct double immunofluorescence staining with proliferation markers (Ki-67) and differentiation markers to contextualize GTF2H1 expression patterns within the cellular state of different tissue regions.

How can researchers differentiate between true GTF2H1 expression changes and technical artifacts in immunoblotting experiments?

Differentiating between true GTF2H1 expression changes and technical artifacts in immunoblotting experiments requires rigorous controls and methodological considerations. Researchers should implement loading controls using housekeeping proteins (e.g., GAPDH, β-actin) alongside normalization to total protein via Ponceau S or REVERT total protein stains, which are less susceptible to experimental conditions. When studying nuclear proteins like GTF2H1, nuclear-specific loading controls such as Lamin B1 or Histone H3 provide more accurate normalization than whole-cell housekeeping proteins . To control for antibody specificity, researchers should include positive control lysates from cells with confirmed GTF2H1 expression and negative controls using siRNA-mediated knockdown samples. Quantification should employ digital imaging systems with dynamic range verification to ensure measurements fall within the linear detection range. Technical replicates (multiple lanes of the same sample) and biological replicates (independent sample preparations) should be included to assess reproducibility. When comparing expression across different conditions, researchers should process all samples simultaneously on the same gel and transfer membrane to eliminate inter-blot variability. For verification of observed changes, researchers should confirm results with alternative GTF2H1 antibodies targeting different epitopes. Finally, orthogonal techniques such as qRT-PCR for GTF2H1 mRNA levels or immunofluorescence can provide complementary evidence to distinguish true biological changes from technical artifacts .

How can researchers investigate the role of GTF2H1 in transcription-coupled repair versus global genomic repair pathways?

Investigating GTF2H1's differential roles in transcription-coupled repair (TCR) versus global genomic repair (GGR) requires sophisticated experimental approaches that can distinguish between these pathways. Researchers should begin by establishing cell models with selective inactivation of TCR or GGR components—for example, using CSB-deficient cells (TCR-defective) or XPC-deficient cells (GGR-defective) with concurrent GTF2H1 modulation via siRNA or overexpression systems . DNA damage can be introduced using transcription-blocking lesions such as cisplatin-induced crosslinks or UV-induced cyclobutane pyrimidine dimers, followed by repair kinetics assessment using lesion-specific antibodies or techniques like ELISA. For pathway-specific analysis, researchers can perform ChIP-seq with GTF2H1 antibodies in cells treated with UV radiation, comparing GTF2H1 recruitment patterns to transcriptionally active versus inactive regions to distinguish TCR from GGR. RNA polymerase II elongation inhibitors like α-amanitin or DRB can be used to block TCR while preserving GGR, allowing researchers to assess GTF2H1's contribution specifically to GGR . Conversely, strand-specific repair assays can quantify TCR efficiency by measuring repair rates in transcribed versus non-transcribed strands of active genes following GTF2H1 modulation. Advanced techniques like DRIP-seq (DNA-RNA Immunoprecipitation followed by sequencing) can identify R-loops that form at stalled transcription sites, potentially revealing how GTF2H1 contributes to resolving these structures during TCR.

What experimental approaches can assess the impact of GTF2H1 post-translational modifications on its function?

Assessing the impact of GTF2H1 post-translational modifications (PTMs) on its function requires multi-dimensional experimental approaches. Researchers should first identify potential PTMs through mass spectrometry analysis of immunoprecipitated native GTF2H1 from cells under various conditions (normal, UV-stressed, serum-starved). Once specific modification sites are identified, site-directed mutagenesis can be employed to generate non-modifiable mutants (e.g., lysine-to-arginine for ubiquitination sites, serine/threonine-to-alanine for phosphorylation sites) or phosphomimetic mutants (serine/threonine-to-glutamate). These mutant constructs should be introduced into GTF2H1-depleted backgrounds to assess functional rescue in transcription assays (using EU incorporation) and repair capacity measurements (using UDS assays) . For temporal dynamics, researchers can combine synchronization techniques with immunoblotting using modification-specific antibodies to track how GTF2H1 modifications change throughout the cell cycle or following DNA damage. Proximity ligation assays can reveal how specific PTMs affect GTF2H1's interactions with other TFIIH components or repair factors. To connect modifications to upstream signaling, researchers can utilize specific kinase, phosphatase, or E3 ligase inhibitors while monitoring GTF2H1 function. For in-depth structural impact assessment, hydrogen-deuterium exchange mass spectrometry can be performed on wild-type versus modified GTF2H1 to determine how PTMs alter protein conformation and potentially expose or conceal interaction surfaces. Finally, CRISPR-Cas9 genome editing to introduce specific PTM site mutations at the endogenous locus will provide the most physiologically relevant assessment of how these modifications affect GTF2H1 function in transcription and repair pathways.

How can single-cell approaches be integrated with GTF2H1 antibody technology to study heterogeneity in transcription and repair capacities?

Integrating single-cell approaches with GTF2H1 antibody technology creates powerful experimental frameworks for studying heterogeneity in transcription and repair capacities across cell populations. Researchers can implement single-cell Western blotting techniques using microfluidic platforms to quantify GTF2H1 protein levels in individual cells, correlating this with functional readouts such as γH2AX foci (DNA damage markers) or EU incorporation (transcriptional activity) . Flow cytometry-based approaches can simultaneously assess GTF2H1 levels via intracellular staining alongside functional markers, enabling high-throughput analysis of thousands of cells. For deeper characterization, researchers can employ CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) by conjugating GTF2H1 antibodies to DNA barcodes, allowing simultaneous measurement of GTF2H1 protein levels and transcriptome-wide gene expression in single cells . Single-cell CUT&Tag using GTF2H1 antibodies can map its genomic binding locations in individual cells, revealing cell-to-cell variation in GTF2H1 chromatin association patterns. For spatial context in tissues, multiplexed immunofluorescence combining GTF2H1 antibodies with markers for cell cycle, differentiation status, and DNA damage can be analyzed using imaging mass cytometry or multiplexed ion beam imaging. Advanced live-cell imaging approaches using GTF2H1 antibody fragments converted to nanobodies and fused to fluorescent proteins can track real-time dynamics of GTF2H1 recruitment to damage sites in individual cells following microirradiation. These integrated approaches enable researchers to connect GTF2H1 expression heterogeneity with functional consequences in transcription regulation and DNA repair capacity at unprecedented resolution .

What strategies can be employed to develop monoclonal antibodies that distinguish between GTF2H1 in its free form versus TFIIH complex-bound form?

Developing monoclonal antibodies that distinguish between free and TFIIH complex-bound GTF2H1 requires strategic epitope selection and screening methodologies. Researchers should begin with structural analysis of GTF2H1 in isolation and within the TFIIH complex using available cryo-EM data to identify regions that undergo conformational changes or become masked upon complex formation. These regions should be used to design peptide immunogens or recombinant protein fragments that represent either the free-specific or complex-specific conformations . Following hybridoma generation, researchers should implement a differential screening cascade that first selects antibodies recognizing GTF2H1, then counter-screens against either purified TFIIH complexes or GTF2H1 alone to identify clones with the desired selectivity profile. Epitope binning experiments using surface plasmon resonance can group antibodies based on their binding regions. For validation, researchers should perform immunoprecipitation experiments under native conditions versus denaturing conditions that disrupt the TFIIH complex, confirming that conformation-specific antibodies only recognize their target under the appropriate conditions. Proximity ligation assays combining the new antibodies with antibodies against other TFIIH components can verify their specificity in cellular contexts. For functional validation, researchers can track the behavior of these antibodies during cellular processes that alter the balance of free versus complex-bound GTF2H1, such as UV irradiation or transcriptional inhibition . Finally, the most promising antibody candidates should be tested in various applications including immunofluorescence microscopy, ChIP-seq, and flow cytometry to determine their utility in distinguishing the different functional pools of GTF2H1 in diverse experimental settings.

How can researchers effectively combine GTF2H1 antibody staining with unscheduled DNA synthesis assays to assess repair capacity?

Effective combination of GTF2H1 antibody staining with unscheduled DNA synthesis (UDS) assays requires careful experimental design to maintain sensitivity of both assays while allowing for their correlation at the single-cell level. Researchers should begin by growing cells on gridded coverslips to enable cell tracking between imaging sessions. Following UV irradiation (typically 10-20 J/m²), cells should be incubated with 5-ethynyl-2'-deoxyuridine (EdU) for 1-3 hours in the presence of hydroxyurea to suppress replicative DNA synthesis . After EdU incorporation, cells should be fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The staining protocol should start with the Click-iT reaction to detect EdU incorporation, using azide-conjugated fluorophores (preferably far-red dyes like Alexa Fluor 647) to minimize spectral overlap with subsequent immunofluorescence. Following the Click-iT reaction, blocking should be performed with 5% normal serum for 1 hour, followed by GTF2H1 antibody incubation at 1:100-1:200 dilution overnight at 4°C . A spectrally compatible secondary antibody (e.g., Alexa Fluor 488) should be used for detection. DAPI counterstaining allows identification of nuclei for automated image analysis. Using high-content imaging systems, researchers can quantify both nuclear GTF2H1 intensity and EdU incorporation at the single-cell level, enabling direct correlation between GTF2H1 expression and repair capacity. This approach can be extended to include additional markers such as CPD-specific antibodies to track both the initial damage and its repair in relation to GTF2H1 levels .

What are the critical considerations when investigating GTF2H1 interactions with XPG using antibody-based approaches?

Investigating GTF2H1 interactions with XPG using antibody-based approaches requires careful attention to several critical factors. First, researchers must select antibodies with epitopes that do not interfere with the interaction interface between GTF2H1 and XPG, which involves the pleckstrin homology domain of GTF2H1 . For co-immunoprecipitation studies, native lysis conditions using buffers containing 0.1-0.5% NP-40 or digitonin rather than stronger detergents like SDS are essential to preserve protein-protein interactions. Additionally, researchers should include DNase I treatment in immunoprecipitation protocols to ensure that observed associations are not DNA-mediated. Proximity ligation assays (PLA) offer an alternative approach to visualize endogenous GTF2H1-XPG interactions in situ, requiring careful antibody pair selection from different host species and extensive controls including single-antibody conditions. For examining how DNA damage affects these interactions, researchers should employ UV irradiation through micropore filters to create localized damage sites, allowing visualization of GTF2H1 and XPG co-recruitment using immunofluorescence . To assess the functional consequences of this interaction, researchers can employ XPG mutants that specifically disrupt GTF2H1 binding without affecting nuclease activity, followed by repair capacity assessment using UDS or lesion removal assays. Time-course studies are crucial, as the GTF2H1-XPG interaction may be transient and stage-specific during repair. Finally, researchers should consider how other TFIIH components influence this interaction, potentially by performing sequential immunoprecipitation (GTF2H1 followed by XPG) to isolate complete complexes versus subcomplexes.