GTF3C4 Antibody

What is GTF3C4 and what is its biological function?

GTF3C4 (General Transcription Factor IIIC Subunit 4) is a protein coding gene essential for RNA polymerase III-mediated transcription of small nuclear and cytoplasmic RNAs, including 5S RNA, tRNA, and adenovirus-associated (VA) RNA. It possesses histone acetyltransferase (HAT) activity with unique specificity for free and nucleosomal H3 . GTF3C4 is also known as TFIIIC90 or KAT12 and functions as part of the transcription factor TFIIIC complex .

The protein cooperates with GTF3C5 in facilitating the recruitment of TFIIIB and RNA polymerase through direct interactions with BRF1, POLR3C, and POLR3F . Subcellularly, GTF3C4 is primarily localized in the nucleoplasm and mitochondrion . Its gene expression and transcriptional control functions make it an important target for research in RNA biology and gene regulation mechanisms.

What are the key specifications to consider when selecting GTF3C4 antibodies?

When selecting GTF3C4 antibodies, researchers should consider several critical specifications:

When selecting an antibody, match these specifications to your experimental requirements, prioritizing antibodies with validation data for your specific application and model system .

How should GTF3C4 antibodies be optimized for Western blot analysis?

Optimizing GTF3C4 antibodies for Western blot requires systematic protocol adjustment:

• Sample Preparation:

  • Extract proteins under denaturing conditions with protease inhibitors

  • Load 15-30 μg total protein per lane (validated in HeLa cells)

  • Include appropriate positive control (e.g., HeLa or U87-MG cells)

• Electrophoresis and Transfer:

  • Use 7.5% SDS-PAGE for optimal separation around 92 kDa (GTF3C4's MW)

  • Ensure complete transfer to membrane using standard transfer conditions

• Antibody Incubation and Detection:

  • Start with manufacturer's recommended dilution (typically 1:500-1:2000)

  • Create a dilution series for optimization (e.g., 1:500, 1:1000, 1:2000)

  • Block with 3-5% non-fat dry milk or BSA in TBST

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly with TBST

  • Use appropriate HRP-conjugated secondary antibody (typically 1:10,000)

• Expected Results:

  • A clear band at approximately 92 kDa corresponding to GTF3C4

  • Minimal background or non-specific bands

  • Increased signal in overexpression studies and reduced/absent signal in knockdown studies

• Troubleshooting:

  • High background: Increase blocking time, reduce antibody concentration

  • Weak signal: Increase antibody concentration, extend exposure time

  • Multiple bands: Verify if they represent isoforms, degradation products, or non-specific binding

The Proteintech antibody (17653-1-AP) and AbClonal antibody (A9287) show clear bands at the expected molecular weight in Western blot analysis of HeLa cells .

What are the optimal conditions for immunohistochemical detection of GTF3C4?

For successful immunohistochemical detection of GTF3C4 in paraffin-embedded tissues:

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) with 1mM EDTA in 10mM Tris buffer (pH 8.5) at 120°C for 3 minutes shows excellent results

  • Alternative: Citrate buffer (pH 6.0) may be used for some antibodies

• Antibody Selection and Dilution:

  • Monoclonal antibodies (e.g., clone OTI4A1) work well at 1:500 dilution

  • Polyclonal antibodies typically used at 1:50-1:500 dilution

  • Test multiple dilutions to determine optimal signal-to-noise ratio

• Tissue Samples with Validated Detection:

  • Human breast cancer tissue shows clear GTF3C4 staining

  • Other validated tissues include:

    • Normal and carcinoma pancreatic tissue

    • Normal and carcinoma lung tissue

    • Normal and carcinoma gastric tissue

    • Carcinoma thyroid tissue

    • Carcinoma liver tissue

• Incubation and Detection System:

  • Primary antibody incubation: 1 hour at room temperature or overnight at 4°C

  • Detection using standard HRP-polymer and DAB chromogen systems

  • Counterstain with hematoxylin for nuclear contrast

• Controls and Interpretation:

  • Include positive control tissue (e.g., breast cancer)

  • Include negative control (primary antibody omitted)

  • GTF3C4 typically shows nuclear and some cytoplasmic staining

  • Compare staining intensity between normal and pathological samples

The above conditions have been validated for multiple GTF3C4 antibodies and provide reliable detection in diverse tissue types .

How can I effectively use GTF3C4 antibodies for immunofluorescence studies?

For optimal immunofluorescence detection of GTF3C4:

• Cell Preparation:

  • Validated cell lines include HeLa, C6, and U-2 OS cells

  • Fix cells with 10% formaldehyde (paraformaldehyde) for 10-15 minutes

  • Alternatively, methanol fixation can be tested if formaldehyde yields high background

• Permeabilization and Blocking:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS

  • Block with 1-5% BSA or normal serum from the same species as the secondary antibody

  • Include 0.1% Tween-20 in blocking buffer to reduce background

• Antibody Incubation:

  • Dilute primary GTF3C4 antibodies at 1:20-1:200 (start with 1:50)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG)

  • Include DAPI for nuclear counterstaining

• Expected Results and Analysis:

  • GTF3C4 shows predominantly nuclear localization

  • Some cytoplasmic staining may be observed depending on cell type

  • Analyze using confocal or fluorescence microscopy

  • Quantify nuclear vs. cytoplasmic signal intensity if needed

• Troubleshooting:

  • High background: Increase blocking time, use more stringent washing

  • Weak signal: Increase antibody concentration, extend incubation time

  • Non-specific binding: Pre-adsorb antibody, optimize blocking conditions

The AbClonal GTF3C4 antibody (A9287) has been successfully used for immunofluorescence in both C6 rat cells and U-2 OS human cells at 1:100 dilution, showing clear nuclear localization .

How can I use GTF3C4 antibodies to study transcriptional complexes?

To investigate GTF3C4's role in transcriptional complexes:

• Co-Immunoprecipitation (Co-IP):

  • Select GTF3C4 antibodies validated for IP (e.g., Proteintech 17653-1-AP at 0.5-4.0 μg per 1-3 mg lysate)

  • Prepare nuclear extracts under native conditions

  • Conduct IP with GTF3C4 antibody and protein A/G beads

  • Analyze co-precipitated proteins by Western blot using antibodies against:

    • Other TFIIIC components (GTF3C1-6)

    • TFIIIB components (BRF1, BDP1, TBP)

    • RNA polymerase III subunits (POLR3C, POLR3F)

  • Include appropriate controls (IgG, input samples)

• Chromatin Immunoprecipitation (ChIP):

  • Use ChIP-certified GTF3C4 antibodies when available

  • Fix cells with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with GTF3C4 antibody

  • Analyze by qPCR targeting known binding sites:

    • tRNA genes

    • 5S rRNA genes

    • Other RNA polymerase III transcribed genes

  • For genome-wide analysis, perform ChIP-seq

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize cells

  • Co-incubate with GTF3C4 antibody and antibody against potential interactor

  • Apply PLA probes, ligate, and amplify

  • Visualize interactions as fluorescent spots

  • Quantify interaction frequency

• Sequential ChIP:

  • Perform first ChIP with GTF3C4 antibody

  • Elute complexes under mild conditions

  • Perform second ChIP with antibodies against other components

  • Analyze co-occupancy at specific genomic loci

These approaches allow investigation of GTF3C4's associations with transcriptional machinery and its genomic binding sites, providing insights into the assembly and function of RNA polymerase III transcription complexes.

How can I validate the specificity of GTF3C4 antibodies in my experimental system?

Rigorous validation of GTF3C4 antibody specificity is crucial for reliable research results:

• Genetic Validation Approaches:

  • siRNA/shRNA Knockdown:

    • Transfect cells with GTF3C4-specific or control siRNA/shRNA

    • Confirm knockdown by RT-qPCR

    • Perform Western blot with GTF3C4 antibody

    • Specific signal should decrease proportionally to knockdown efficiency

  • CRISPR/Cas9 Knockout:

    • Generate GTF3C4 knockout cell lines

    • Confirm knockout by genomic sequencing and RT-qPCR

    • The specific band should be absent in Western blots of knockout samples

  • Overexpression:

    • Transfect cells with GTF3C4 expression vector vs. empty vector

    • Specific signal should increase in overexpressing cells

• Biochemical Validation:

  • Peptide Competition:

    • Pre-incubate antibody with excess immunizing peptide

    • Run parallel assays with blocked and unblocked antibody

    • Specific signal should be eliminated or significantly reduced

  • Immunoprecipitation-Mass Spectrometry:

    • Perform IP with GTF3C4 antibody

    • Analyze by mass spectrometry

    • GTF3C4 should be among the most abundant proteins identified

• Cross-Validation with Multiple Antibodies:

  • Test multiple GTF3C4 antibodies targeting different epitopes

  • Compare staining patterns across applications

  • Consistent results with antibodies against different epitopes increase confidence

• Controls for Specific Applications:

  • Western Blot: Include lysates from cells with known GTF3C4 expression (e.g., HeLa cells)

  • IHC/IF: Include positive control tissues/cells and negative controls (primary antibody omitted)

  • ChIP: Include IgG control and input samples

This multi-faceted validation approach ensures antibody specificity and reliability across experimental systems.

What strategies can help investigate GTF3C4's histone acetyltransferase activity?

To study GTF3C4's histone acetyltransferase (HAT) activity:

• In Vitro HAT Assay with Immunoprecipitated GTF3C4:

  • Immunoprecipitate GTF3C4 using validated antibodies (e.g., Proteintech 17653-1-AP)

  • Incubate with:

    • Purified histone H3 or nucleosomes

    • Acetyl-CoA (radiolabeled or unlabeled)

    • HAT assay buffer

  • Detect acetylation by:

    • Western blot with anti-acetyl-H3 antibodies

    • Fluorography (for radiolabeled assays)

    • Mass spectrometry to identify specific acetylation sites

  • Controls:

    • Known HAT enzyme (positive control)

    • IP with non-specific IgG (negative control)

• Cell-Based Acetylation Analysis:

  • Manipulate GTF3C4 levels via:

    • Overexpression of wild-type or HAT-dead mutant

    • siRNA/shRNA knockdown

    • CRISPR/Cas9 knockout

  • Extract histones using acid extraction

  • Analyze H3 acetylation levels by Western blot

  • Quantify acetylation relative to total H3

• ChIP-seq Correlation Analysis:

  • Perform parallel ChIP-seq with:

    • GTF3C4 antibody

    • Anti-acetylated histone H3 antibody

  • Analyze overlap between GTF3C4 binding sites and acetylated regions

  • Compare acetylation patterns in wild-type and GTF3C4-depleted cells

• Domain-Specific Investigations:

  • Generate domain deletion constructs of GTF3C4

  • Focus on regions critical for HAT activity

  • Use antibodies targeting regions outside HAT domain

  • Examine changes in histone acetylation

• Acetylation Target Identification:

  • Combine GTF3C4 IP with acetylome analysis

  • Identify differential acetylation patterns upon GTF3C4 manipulation

  • Validate using site-specific acetylation antibodies

These approaches provide complementary strategies to characterize GTF3C4's HAT activity, its regulatory mechanisms, and its significance in transcriptional regulation.

How can I resolve non-specific binding issues with GTF3C4 antibodies?

Non-specific binding can significantly impact GTF3C4 antibody performance. Here are methodological solutions for different applications:

• Western Blot Troubleshooting:

  • High Background Across Membrane:

    • Increase blocking time/concentration (use 5% non-fat milk or BSA)

    • Add 0.1-0.5% Tween-20 to washing buffers

    • Decrease antibody concentration (try 1:2000-1:2400 for primary)

    • Use fresher transfer buffer and ensure adequate transfer time

  • Multiple Bands:

    • Verify if they represent GTF3C4 isoforms or degradation products

    • Use freshly prepared samples with protease inhibitors

    • Try different antibodies targeting different epitopes

    • Perform knockdown experiments to identify specific bands

• Immunohistochemistry Optimization:

  • High Background:

    • Optimize antigen retrieval (EDTA buffer pH 9.0 works well for many GTF3C4 antibodies)

    • Include endogenous peroxidase and protein blocking steps

    • Use species-specific blocking serum

    • Reduce antibody concentration (try 1:250-1:500)

    • Add additional washing steps

  • Non-specific Staining:

    • Pre-adsorb antibody with tissue powder

    • Increase antibody dilution

    • Optimize incubation time and temperature

• Immunofluorescence Refinement:

  • High Background:

    • Use more stringent blocking (5-10% normal serum)

    • Include 0.1-0.3% Triton X-100 in buffers

    • Add 0.1-0.5M NaCl to washing buffers to increase stringency

    • Optimize fixation conditions

    • Try shorter antibody incubation times

  • Autobuorescence:

    • Include autofluorescence quenching steps

    • Adjust microscope settings to minimize background

• Cross-Application Strategies:

  • Antibody Validation:

    • Test multiple GTF3C4 antibodies targeting different epitopes

    • Use well-characterized positive controls (HeLa cells show reliable GTF3C4 expression)

    • Include appropriate negative controls in each experiment

These systematic troubleshooting approaches address the most common issues with GTF3C4 antibodies across different applications.

What are the critical factors for successfully immunoprecipitating GTF3C4?

Successful immunoprecipitation (IP) of GTF3C4 requires attention to several critical factors:

• Antibody Selection:

  • Use antibodies validated for IP applications

  • Proteintech 17653-1-AP has been validated for GTF3C4 IP at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Consider the epitope location - antibodies targeting accessible epitopes perform better

• Cell Lysis and Extract Preparation:

  • Lysis Buffer Composition:

    • For protein-protein interaction studies: Non-denaturing buffer (e.g., 150mM NaCl, 50mM Tris pH 7.5, 1% NP-40)

    • For strict specificity: RIPA buffer (more stringent)

    • Include protease inhibitors, phosphatase inhibitors, and EDTA

  • Lysis Conditions:

    • Optimize cell/tissue disruption method

    • Keep samples cold throughout processing

    • Clear lysates by centrifugation (12,000-14,000 rpm, 10 minutes, 4°C)

• Pre-clearing and Blocking:

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

  • Block beads with BSA or non-fat milk to minimize background

  • Consider pre-adsorption with irrelevant IgG for highly specific IP

• Antibody Binding Conditions:

  • Amount Optimization:

    • Start with 2-5 μg antibody per 1 mg of lysate

    • Adjust based on GTF3C4 abundance in your sample

  • Incubation Parameters:

    • Incubate overnight at 4°C with gentle rotation

    • Avoid harsh mixing that might disrupt protein complexes

• Washing and Elution:

  • Washing Stringency:

    • For protein complexes: Gentle washing with lysis buffer

    • For high specificity: Include higher salt (up to 300mM NaCl) in later washes

    • Typical protocol: 3-5 washes, 5 minutes each at 4°C

  • Elution Methods:

    • Denaturing: SDS-PAGE sample buffer at 95°C

    • Native: Peptide competition or low pH elution

• Controls and Validation:

  • Include IP with non-specific IgG from the same species

  • Include input sample (5-10% of starting material)

  • Verify GTF3C4 enrichment by Western blot

  • For novel interactions, validate with reverse IP

Following these guidelines increases the likelihood of successful GTF3C4 immunoprecipitation while minimizing non-specific interactions.

How do monoclonal and polyclonal GTF3C4 antibodies compare in performance?

Monoclonal and polyclonal GTF3C4 antibodies each offer distinct advantages for different research applications:

Research-Based Recommendations:

• For Western Blot: Both types perform well, but monoclonals provide cleaner backgrounds and more consistent results .

• For Immunohistochemistry:

  • Monoclonal antibodies (e.g., OTI4A1) show excellent staining in multiple tissues including pancreas, lung, thyroid, and gastric tissues .

  • Polyclonal antibodies also perform well in IHC applications with proper optimization .

• For Immunofluorescence:

  • Polyclonal antibodies typically show stronger nuclear staining of GTF3C4 .

  • Dilution optimization is particularly important (1:50-1:200 range).

• For Immunoprecipitation:

  • Polyclonal antibodies often perform better due to multiple epitope recognition .

  • Critical for protein complex and interaction studies.

• For ChIP Applications:

  • Specifically ChIP-validated antibodies are recommended .

  • Epitope accessibility in chromatin context is critical.

This comparative analysis provides a framework for selecting the most appropriate GTF3C4 antibody type based on specific experimental requirements and technical considerations.

What are the key differences in epitope targeting among available GTF3C4 antibodies?

Understanding epitope differences among GTF3C4 antibodies is crucial for experimental design:

Functional and Structural Considerations:

• Domain-Specific Research:

  • N-terminal antibodies (AA 73-334): Target regions containing portions of GTF3C4's RNA polymerase III interaction domains

  • C-terminal antibodies (AA 583-822): Target regions containing part of the histone acetyltransferase domain

  • Selecting antibodies targeting specific functional domains depends on research objectives

• Isoform Detection:

  • N-terminal antibodies may detect all potential isoforms

  • C-terminal antibodies might miss truncated forms

  • Consider known or potential isoforms in your experimental system

• Post-Translational Modifications:

  • Antibodies targeting heavily modified regions may show variable binding

  • Epitopes containing potential phosphorylation, acetylation, or ubiquitination sites may affect antibody recognition

• Protein-Protein Interactions:

  • For co-IP studies, choose antibodies with epitopes outside interaction domains

  • C-terminal antibodies may be preferable for studying N-terminal interactions

  • N-terminal antibodies may work better for capturing C-terminal interaction partners

• Cross-Application Compatibility:

  • Some epitopes perform better in native conditions (IP, IF)

  • Others work optimally in denatured states (WB)

  • Proteintech 17653-1-AP (AA 473-822) shows versatility across multiple applications

Understanding these epitope differences allows researchers to select GTF3C4 antibodies aligned with their specific experimental objectives and biological questions.

How are GTF3C4 antibodies used in cancer research applications?

GTF3C4 antibodies have demonstrated utility in cancer research through multiple approaches:

• Expression Analysis in Cancer Tissues:

  • IHC studies reveal GTF3C4 expression across various cancer types:

    • Breast cancer tissue shows clear GTF3C4 immunoreactivity

    • Lung carcinoma exhibits GTF3C4 expression

    • Pancreatic carcinoma shows GTF3C4 immunostaining

    • Gastric carcinoma demonstrates GTF3C4 expression

    • Thyroid carcinoma and liver carcinoma also show GTF3C4 expression

  • Comparative expression studies between cancerous and normal tissues provide insights into potential functional roles

• Methodological Approaches in Cancer Cell Lines:

  • Western blot analysis of cancer cell line panels:

    • Multiple cancer cell lines show consistent GTF3C4 expression

    • U87-MG glioblastoma cells serve as positive controls

  • Immunofluorescence in cancer cell models:

    • HeLa cervical cancer cells show nuclear GTF3C4 localization

    • U-2 OS osteosarcoma cells exhibit clear nuclear staining

• Applications in Transcriptional Dysregulation Studies:

  • Investigating RNA polymerase III activity in cancer:

    • GTF3C4 antibodies enable analysis of TFIIIC complex assembly

    • ChIP approaches allow mapping of GTF3C4 binding to tRNA and 5S rRNA genes

  • Histone acetylation studies:

    • GTF3C4's HAT activity may contribute to cancer-associated epigenetic alterations

    • Antibodies facilitate investigation of this potential mechanism

• Protein-Protein Interaction Networks:

  • Co-IP with GTF3C4 antibodies to identify:

    • Cancer-specific protein interactions

    • Altered complex formation in malignant states

  • Sequential ChIP to identify co-occupancy with other cancer-relevant factors

• Technical Considerations for Cancer Research:

  • Tissue-specific optimization:

    • EDTA-based antigen retrieval (pH 8.5-9.0) works well for most cancer tissues

    • Dilution optimization may vary by cancer type (typically 1:100-1:500)

  • Controls:

    • Include known positive cancer tissues

    • Compare with corresponding normal tissues when possible

These applications demonstrate how GTF3C4 antibodies enable investigation of this transcription factor's potential roles in cancer biology, particularly in relation to RNA polymerase III activity and epigenetic regulation.

What experimental considerations are important when using GTF3C4 antibodies in multiplex analysis?

When incorporating GTF3C4 antibodies into multiplex analysis protocols:

• Antibody Compatibility Planning:

  • Host Species Selection:

    • Choose GTF3C4 antibodies from different host species than other target antibodies

    • Available options include rabbit polyclonal/monoclonal and mouse monoclonal GTF3C4 antibodies

    • Example combination: Rabbit anti-GTF3C4 with mouse antibodies against other targets

  • Isotype Considerations:

    • Note isotype information (IgG, IgG1, etc.) for secondary antibody selection

    • Mouse monoclonal GTF3C4 antibodies are typically IgG1 isotype

    • Rabbit polyclonal antibodies are generally IgG

• Fluorescent Multiplex Immunofluorescence:

  • Direct Conjugation Options:

    • Consider Alexa Fluor 750-conjugated GTF3C4 antibody for multiplex IF

    • Select non-overlapping fluorophores for other targets

  • Sequential Staining Protocol:

    • Optimize individual antibodies before multiplexing

    • Consider tyramide signal amplification for weak signals

    • Test antibody combinations for cross-reactivity

    • Include single-stain controls for spectral unmixing

• Multiplex Immunohistochemistry:

  • Chromogenic Multiplexing:

    • Use different enzyme-substrate combinations (HRP, AP)

    • Optimize antibody dilutions for each target (GTF3C4 typically 1:100-1:500)

    • Consider sequential staining with stripping or blocking between rounds

  • Blocking Considerations:

    • Complete blocking between sequential staining rounds

    • Use species-specific secondary antibody blockers

    • Consider avidin/biotin blocking when using biotinylated secondaries

• Flow Cytometry Applications:

  • Protocol Optimization:

    • Fixation and permeabilization are crucial for intracellular GTF3C4 detection

    • Test different permeabilization reagents (saponin, Triton X-100)

    • Consider fluorophore brightness hierarchies (assign brightest fluorophores to lowest expression targets)

  • Controls:

    • Include fluorescence-minus-one (FMO) controls

    • Use isotype controls for each antibody class

    • Consider GTF3C4 knockdown cells as biological controls

• Data Analysis Considerations:

  • Colocalization Analysis:

    • Use appropriate colocalization algorithms (Pearson's, Manders')

    • Analyze GTF3C4 colocalization with other nuclear proteins

    • Control for random overlap in dense nuclear regions

  • Quantitative Assessment:

    • Standardize image acquisition parameters

    • Develop consistent quantification methods

    • Consider machine learning approaches for complex pattern recognition

Careful consideration of these factors enables successful incorporation of GTF3C4 antibodies into multiplex analysis workflows while minimizing technical artifacts and cross-reactivity issues.