GTF3C6 Antibody

What is GTF3C6 and what cellular functions does it perform?

GTF3C6 (General Transcription Factor IIIC, Polypeptide 6, alpha 35kDa) is a component of the transcription factor IIIC complex. This protein is involved in RNA polymerase III-mediated transcription, which is responsible for synthesizing small RNAs like tRNAs and 5S rRNA. GTF3C6 is also known by alternative names including C6orf51, TFIIIC35, and bA397G5.3 . The protein has an observed molecular weight of approximately 35-38 kDa . In terms of structure, GTF3C6 contains sequences that enable it to interact with DNA and other proteins within the transcription complex. Understanding its function is essential for research into basic transcriptional mechanisms and potentially for investigating certain disease states where RNA polymerase III function may be dysregulated.

What are the validated applications for GTF3C6 antibodies?

GTF3C6 antibodies have been validated for multiple laboratory applications. According to validation data, these antibodies can be reliably used in:

• Western Blot (WB) - Validated at dilutions ranging from 1:500 to 1:6000

• Immunohistochemistry (IHC) - Validated at dilutions of 1:500-1:2000

• Immunoprecipitation (IP) - Using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• ELISA - Various dilutions depending on the specific antibody formulation

Some antibodies are also available in conjugated forms, including biotin-conjugated and FITC-conjugated versions, which expand their utility for fluorescence-based detection methods .

What species reactivity has been confirmed for GTF3C6 antibodies?

Current GTF3C6 antibodies have been tested and confirmed to react with:

• Human samples - All suppliers confirm human reactivity

• Mouse samples - Some antibodies, such as Proteintech's 26382-1-AP, show cross-reactivity with mouse tissues

It's important to note that species reactivity varies between manufacturers and individual antibody products. Researchers should verify the specific reactivity profile before selecting an antibody for their target species .

What is the recommended sample preparation protocol for Western blot using GTF3C6 antibodies?

For optimal Western blot results with GTF3C6 antibodies, follow these methodological steps:

• Prepare cell or tissue lysates using a complete protease inhibitor cocktail

• Determine protein concentration and load 20-40 μg of total protein per lane

• Separate proteins using SDS-PAGE (10-12% gel recommended based on the 35-38 kDa molecular weight of GTF3C6)

• Transfer proteins to PVDF or nitrocellulose membrane

• Block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature

• Incubate with primary GTF3C6 antibody at the appropriate dilution (1:1000-1:6000 recommended for WB)

• Wash membrane 3-5 times with TBST

• Incubate with HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence detection

The antibody has been successfully tested on various cell lines including HeLa, HepG2, and K-562 cells .

What are the proper storage conditions for GTF3C6 antibodies?

To maintain antibody activity and extend shelf life, GTF3C6 antibodies should be stored according to the following guidelines:

• Store at -20°C for long-term storage

• For antibodies in liquid formulation with preservatives (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3), aliquoting is not necessary for -20°C storage

• Avoid repeated freeze-thaw cycles, which can lead to denaturation and decreased activity

• For working solutions, store at 4°C for up to one month

• Some formulations (particularly the smaller 20μL sizes) may contain 0.1% BSA as a stabilizer

The antibodies are generally stable for one year after shipment when stored properly .

How can I optimize GTF3C6 antibody for immunoprecipitation experiments?

Optimizing GTF3C6 antibody for immunoprecipitation requires careful consideration of several experimental variables:

• Antibody amount: Use 0.5-4.0 μg of GTF3C6 antibody per 1.0-3.0 mg of total protein lysate . Begin with the middle of this range and adjust based on results.

• Lysis buffer selection: Use a buffer that preserves protein-protein interactions while efficiently lysing cells. RIPA buffer may be too harsh; consider NP-40 or Triton X-100 based buffers with protease inhibitors.

• Pre-clearing step: To reduce non-specific binding, pre-clear lysates with control IgG and Protein A/G beads for 1 hour at 4°C before adding the GTF3C6 antibody.

• Incubation conditions: For efficient antigen capture, incubate the antibody-lysate mixture overnight at 4°C with gentle rotation.

• Wash optimization: Perform 4-5 washes with decreasing salt concentrations to remove non-specific interactions while preserving specific binding.

• Elution method: Elute the immunoprecipitated complex with either low pH glycine buffer or by boiling in SDS sample buffer, depending on downstream applications.

• Controls: Always include a negative control (normal rabbit IgG) and, if possible, a positive control (lysate from cells known to express high levels of GTF3C6, such as HepG2 cells) .

The protocol can be validated by Western blot analysis of the immunoprecipitated material using another GTF3C6 antibody that recognizes a different epitope.

What are the optimal conditions for antigen retrieval when using GTF3C6 antibody in immunohistochemistry?

Achieving optimal staining with GTF3C6 antibody in immunohistochemistry requires proper antigen retrieval. Based on validation data:

• Recommended method: Heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0 is suggested as the primary approach .

• Alternative method: Citrate buffer at pH 6.0 can be used as an alternative if the primary method yields unsatisfactory results .

• Protocol details:

  • For HIER with TE buffer (pH 9.0): Heat sections in buffer for 15-20 minutes at 95-100°C

  • For citrate buffer (pH 6.0): Heat sections for 10-15 minutes at 95-100°C

  • Allow sections to cool slowly to room temperature before proceeding with blocking step

• Tissue-specific considerations: Different tissue types may require adjusted retrieval times. For example:

  • Human urothelial carcinoma tissue, human lung cancer tissue, and mouse lung tissue have been successfully stained following these retrieval protocols

• Antibody dilution: Use at 1:500-1:2000 dilution for IHC applications after antigen retrieval

• Visualization system: Compatible with both peroxidase/DAB and fluorescent secondary antibody detection systems

Remember that antigen retrieval conditions may need to be optimized for specific tissue types or fixation methods.

How can I validate the specificity of a GTF3C6 antibody for my particular research application?

Validating antibody specificity is crucial for ensuring reliable results. For GTF3C6 antibodies, consider implementing these validation strategies:

• Positive and negative controls:

  • Use cell lines with known GTF3C6 expression levels (HeLa, HepG2, K-562 have been confirmed positive)

  • Include GTF3C6-knockout or knockdown samples as negative controls

  • Compare staining patterns across multiple tissue types

• Molecular weight verification:

  • In Western blot, confirm that the detected band appears at the expected molecular weight of 35-38 kDa

  • Be aware of potential post-translational modifications that might alter the apparent molecular weight

• Multiple antibodies approach:

  • Test multiple GTF3C6 antibodies recognizing different epitopes

  • Compare staining patterns between antibodies from different vendors or clones

• Epitope blocking:

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

  • This should eliminate specific signals in a concentration-dependent manner

• Orthogonal methods:

  • Correlate protein detection with mRNA expression data

  • Confirm localization patterns with tagged overexpression constructs

• Immunoprecipitation-mass spectrometry:

  • Perform IP followed by mass spectrometry to confirm that GTF3C6 is indeed being precipitated

  • Check for known GTF3C6 interaction partners in the precipitate

• Application-specific validation:

  • For IHC: Compare staining patterns with published GTF3C6 expression data

  • For IP: Verify enrichment by Western blot using another GTF3C6 antibody

These validation steps should be documented and reported in publications to support the reliability of experimental findings.

How does GTF3C6 expression vary across different cell lines and tissues?

GTF3C6 expression exhibits tissue and cell type specificity that researchers should consider when designing experiments:

• Cell line expression:

  • Confirmed expression in human cell lines including HeLa (cervical cancer), HepG2 (liver cancer), and K-562 (leukemia) cells

  • Expression levels may vary significantly between cell types, reflecting tissue-specific transcriptional regulation

• Tissue expression:

  • Positive immunohistochemical staining has been validated in:

    • Human urothelial carcinoma tissue

    • Human lung cancer tissue

    • Mouse lung tissue

  • Expression patterns may differ between normal and cancerous tissues of the same origin

• Subcellular localization:

  • As a transcription factor component, GTF3C6 is primarily localized to the nucleus

  • Some cytoplasmic staining may be observed, particularly in certain cancer cell types

  • Precise localization patterns should be verified using subcellular fractionation or co-staining with organelle markers

• Developmental considerations:

  • Expression levels may vary during development and cellular differentiation

  • Consider developmental stage when examining GTF3C6 in embryonic or rapidly dividing tissues

• Disease-associated changes:

  • Expression alterations may occur in certain pathological states

  • Compare expression between normal and disease-state tissues when investigating pathology-related research questions

Understanding tissue-specific expression patterns is essential for experimental design, particularly when selecting appropriate positive control samples and interpreting results in the context of tissue-specific biology.

What are the considerations for using GTF3C6 antibodies in co-immunoprecipitation to study protein interactions?

When using GTF3C6 antibodies for co-immunoprecipitation to investigate protein-protein interactions, consider these methodological aspects:

• Buffer composition:

  • Use gentler lysis buffers (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol)

  • Avoid harsh detergents like SDS that disrupt protein-protein interactions

  • Include protease inhibitors, phosphatase inhibitors, and potentially RNase inhibitors (if RNA-mediated interactions are suspected)

• Antibody selection:

  • Choose antibodies validated for IP applications (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Consider epitope location - antibodies targeting functional domains might disrupt certain protein interactions

• Cross-linking considerations:

  • For transient or weak interactions, consider using reversible cross-linkers like DSP (dithiobis(succinimidyl propionate))

  • Cross-linking can stabilize complexes but may introduce artifacts if not carefully controlled

• Nuclear extraction protocols:

  • Since GTF3C6 is primarily nuclear, optimize nuclear extraction protocols

  • Consider using specialized nuclear complex isolation buffers that preserve transcription factor complexes

• Controls for specificity:

  • Include isotype control antibody (normal rabbit IgG) IP

  • Consider using cells with GTF3C6 knockdown as negative controls

  • Use GTF3C6 overexpression systems as positive controls

• Validation of interactions:

  • Perform reciprocal co-IP (IP with antibody against the interacting protein and blot for GTF3C6)

  • Verify that interactions occur at endogenous expression levels

  • Consider secondary confirmation methods like proximity ligation assay

• Detection strategies:

  • For known interactions, use specific antibodies against suspected binding partners

  • For discovery of novel interactions, consider mass spectrometry analysis of co-immunoprecipitated proteins

When analyzing results, remember that GTF3C6 functions as part of the larger TFIIIC complex in transcription, so co-IP may pull down other components of this complex.

How can I troubleshoot weak or absent signals in Western blots using GTF3C6 antibodies?

When encountering weak or no signal in Western blots with GTF3C6 antibodies, systematically address these potential issues:

• Sample preparation issues:

  • Ensure complete protein extraction, especially for nuclear proteins like GTF3C6

  • Verify protein concentration measurement and consider increasing loading amount

  • Add fresh protease inhibitors to prevent degradation

  • For difficult tissues, optimize lysis buffer composition

• Antibody-specific factors:

  • Adjust antibody dilution - try a more concentrated solution (1:500-1:1000)

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

  • Verify antibody storage conditions and check expiration date

  • Consider testing alternative GTF3C6 antibodies that target different epitopes

• Transfer and detection optimization:

  • Ensure complete protein transfer to membrane (verify with reversible total protein stain)

  • Try different membrane types (PVDF vs. nitrocellulose)

  • Increase blocking stringency if background is high

  • Use more sensitive detection reagents or increase exposure time

  • Consider enhanced chemiluminescence substrates with higher sensitivity

• Protocol modifications:

  • Adjust separation conditions (run longer for better resolution around 35-38 kDa)

  • Try different blocking agents (milk vs. BSA)

  • Extend washing steps to reduce background

  • Consider using a signal enhancer solution before primary antibody incubation

• Expression-related considerations:

  • Confirm GTF3C6 expression in your sample type

  • Use positive control lysates from cells known to express GTF3C6 (HeLa, HepG2, K-562)

  • Consider treatment conditions that might alter GTF3C6 expression

• Technical verifications:

  • Strip and reprobe the membrane with a housekeeping protein antibody

  • Validate the secondary antibody using another primary antibody of the same host species

  • Perform Ponceau S staining to confirm successful protein transfer

Systematic testing of these variables should help identify the source of weak signals and guide appropriate adjustments to your protocol.

What are the considerations for using GTF3C6 antibodies in chromatin immunoprecipitation (ChIP) assays?

While ChIP is not explicitly listed among the validated applications for current GTF3C6 antibodies in the search results, researchers interested in adapting these antibodies for ChIP should consider:

• Antibody suitability assessment:

  • Test antibody specificity in applications like IP and Western blot first

  • Verify that the antibody recognizes the native (non-denatured) form of GTF3C6

  • Choose antibodies that immunoprecipitate GTF3C6 efficiently

• Cross-linking optimization:

  • For transcription factors like GTF3C6, optimize formaldehyde cross-linking (typically 0.75-1% for 10-15 minutes)

  • Consider dual cross-linking approaches (formaldehyde plus protein-protein cross-linkers like DSG)

  • Test variable cross-linking times to find optimal conditions

• Chromatin preparation:

  • Optimize sonication conditions to generate 200-500 bp fragments

  • Ensure complete nuclear lysis to release chromatin-bound GTF3C6

  • Verify sonication efficiency by agarose gel electrophoresis

• Antibody amount optimization:

  • Start with 2-5 μg of antibody per ChIP reaction

  • Scale based on IP efficiency data (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Include excess bead capacity to ensure complete antibody capture

• Buffer modifications:

  • For transcription factors, consider reducing stringency of wash buffers

  • Include protease inhibitors in all buffers

  • Consider adding BSA (0.1-0.5%) to reduce non-specific binding

• Controls and validation:

  • Include IgG control and input samples

  • Validate enrichment at known or predicted GTF3C6 binding sites

  • Consider using GTF3C6 knockdown cells as negative controls

  • Use positive controls like RNA Pol III-transcribed genes

• Data analysis considerations:

  • Focus initial analyses on tRNA genes and other RNA Pol III-transcribed loci

  • Compare GTF3C6 binding with other TFIIIC components

  • Correlate binding with transcriptional activity of target genes

Remember that GTF3C6, as part of the TFIIIC complex, is expected to bind primarily to promoters of genes transcribed by RNA polymerase III, such as tRNA genes and 5S rRNA genes.

How can I quantitatively compare GTF3C6 expression across different experimental conditions?

For accurate quantitative comparison of GTF3C6 expression across experimental conditions, consider these methodological approaches:

• Western blot quantification:

  • Use antibody dilutions within the linear detection range (1:1000-1:6000)

  • Include a concentration gradient of positive control (HepG2 or HeLa lysate)

  • Normalize to appropriate loading controls (β-actin, GAPDH, or total protein stain)

  • Use digital image analysis software to quantify band intensity

  • Perform at least three biological replicates for statistical analysis

• Immunohistochemistry quantification:

  • Use consistent staining protocols across all samples (1:500-1:2000 dilution)

  • Implement digital pathology approaches for objective scoring

  • Consider H-score, Allred score, or percent positive cells for semi-quantitative analysis

  • Use automated image analysis software for more precise quantification

  • Include control tissues on the same slide to account for staining variability

• ELISA-based quantification:

  • Consider developing sandwich ELISA using two different GTF3C6 antibodies

  • Generate standard curves using recombinant GTF3C6 protein

  • Normalize to total protein concentration

  • Account for matrix effects by preparing standards in the same buffer as samples

• Flow cytometry application:

  • For intracellular GTF3C6 detection, use FITC-conjugated antibodies

  • Optimize fixation and permeabilization conditions for nuclear proteins

  • Include fluorescence minus one (FMO) controls

  • Report data as mean fluorescence intensity (MFI) or percent positive cells

• qPCR correlation:

  • Correlate protein expression with mRNA levels

  • Design primers specific to GTF3C6 transcript

  • Consider the relationship between transcript and protein levels may not be linear

• Statistical considerations:

  • Perform appropriate statistical tests based on data distribution

  • Consider power analysis to determine required sample size

  • Report effect sizes alongside p-values

  • Account for multiple comparisons if analyzing many conditions

These approaches provide complementary information and should ideally be used in combination to obtain a comprehensive understanding of GTF3C6 expression changes.

How can I determine if post-translational modifications affect GTF3C6 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition. To determine if PTMs affect GTF3C6 antibody binding:

• Epitope mapping analysis:

  • Identify the specific amino acid sequence recognized by the antibody

  • Review the immunogen sequence (amino acids 1-213 of human GTF3C6 for some antibodies)

  • Analyze this region for potential modification sites using bioinformatics tools

• Multiple antibody comparison:

  • Compare results from antibodies recognizing different epitopes of GTF3C6

  • Discrepancies in detection may indicate modification-sensitive epitopes

  • Use antibodies specifically designed to detect modified forms, if available

• Treatment with modifying/demodifying enzymes:

  • Treat lysates with phosphatases to remove phosphorylation

  • Use deubiquitinating enzymes to remove ubiquitin modifications

  • Compare antibody detection before and after treatment

• Mobility shift analysis:

  • Look for shifts from the expected 35-38 kDa molecular weight

  • Multiple bands may indicate different modification states

  • Compare mobility patterns across different cell types and treatments

• Immunoprecipitation-mass spectrometry:

  • Perform IP with GTF3C6 antibody followed by mass spectrometry

  • Identify PTMs present on the immunoprecipitated protein

  • Correlate modification state with antibody recognition efficiency

• Controlled modification studies:

  • Treat cells with modifiers of specific PTMs (kinase inhibitors, HDAC inhibitors, etc.)

  • Monitor changes in antibody detection following treatment

  • Correlate with functional changes in GTF3C6 activity

• Recombinant protein controls:

  • Compare antibody detection of recombinant unmodified GTF3C6 versus endogenous protein

  • Use site-directed mutagenesis to create modification-mimicking mutants

Understanding how PTMs affect antibody recognition is crucial for accurate interpretation of results, especially when comparing GTF3C6 across different cellular states where modification status may vary.

What control samples should be included when using GTF3C6 antibodies for various applications?

Proper experimental controls are essential for accurate interpretation of results when using GTF3C6 antibodies:

• Western blot controls:

  • Positive control: Lysates from cells with confirmed GTF3C6 expression (HeLa, HepG2, K-562 cells)

  • Negative control: Lysates from cells with GTF3C6 knockdown/knockout

  • Loading control: Housekeeping protein or total protein stain

  • Molecular weight marker: To confirm the expected 35-38 kDa band size

• Immunohistochemistry controls:

  • Positive tissue control: Human urothelial carcinoma, human lung cancer, or mouse lung tissue

  • Negative tissue control: Tissues with minimal GTF3C6 expression

  • Technical negative control: Primary antibody omission

  • Antibody specificity control: Antigen pre-absorption

  • Isotype control: Normal rabbit IgG at the same concentration

• Immunoprecipitation controls:

  • Input sample: Pre-IP lysate (typically 5-10%)

  • Negative control IP: Normal rabbit IgG

  • Supernatant sample: Post-IP supernatant to assess depletion efficiency

  • Specificity control: IP from GTF3C6-depleted cells

• ELISA controls:

  • Standard curve: Recombinant GTF3C6 protein at known concentrations

  • Blank wells: All reagents except primary antibody

  • Negative sample: From cells with minimal GTF3C6 expression

  • Dilution linearity: Serial dilutions of positive samples

• Flow cytometry controls:

  • Unstained cells

  • Isotype control: Rabbit IgG-FITC

  • Single stain controls: For compensation in multi-color panels

  • FMO (Fluorescence Minus One) controls

• Specialized experimental controls:

  • Treatment response: Time course and dose response samples

  • Biological replicates: Multiple independent samples

  • Technical replicates: Repeated measurements of the same sample

Including these controls allows for proper validation of results and helps troubleshoot any issues that may arise during experimentation.

How can I design experiments to study GTF3C6 function using these antibodies?

Designing experiments to study GTF3C6 function requires strategic use of antibodies combined with other molecular biology techniques:

• Expression correlation studies:

  • Use GTF3C6 antibodies in Western blot or IHC to quantify protein levels across:

    • Different cell types or tissues

    • Disease versus normal states

    • Developmental stages

    • Response to various stimuli

  • Correlate protein expression with functional outcomes or phenotypic changes

• Protein-protein interaction network mapping:

  • Use GTF3C6 antibodies for co-immunoprecipitation (0.5-4.0 μg antibody per 1.0-3.0 mg lysate)

  • Identify interaction partners by mass spectrometry

  • Validate specific interactions with reciprocal co-IP

  • Map interaction domains using truncated protein constructs

  • Visualize interactions with proximity ligation assay (PLA)

• Transcriptional regulation studies:

  • Adapt GTF3C6 antibodies for chromatin immunoprecipitation

  • Map binding sites genome-wide with ChIP-seq

  • Correlate binding with RNA Pol III transcriptional output

  • Study the assembly dynamics of TFIIIC complex components

  • Investigate regulation in response to cellular stressors

• Functional perturbation studies:

  • Use antibodies to monitor GTF3C6 levels following:

    • siRNA or shRNA knockdown

    • CRISPR/Cas9 knockout or mutation

    • Overexpression studies

  • Correlate protein level changes with functional outcomes

• Cellular localization studies:

  • Use IHC or immunofluorescence to determine subcellular localization

  • Track localization changes in response to stimuli

  • Perform fractionation followed by Western blot to confirm microscopy findings

• Disease-relevance investigations:

  • Compare GTF3C6 expression in normal versus disease tissues

  • Assess correlation with disease progression or prognosis

  • Investigate expression changes in response to therapeutic interventions

• Post-translational modification analysis:

  • Use antibodies in IP-mass spectrometry workflows

  • Compare GTF3C6 modification states across conditions

  • Correlate modifications with functional changes

These experimental approaches, combined with appropriate controls and validation strategies, can provide comprehensive insights into GTF3C6 function and regulation.

What are the best methods to validate and compare results from different GTF3C6 antibodies?

When validating and comparing results from different GTF3C6 antibodies, implement these methodological approaches:

• Side-by-side comparison:

  • Test multiple antibodies simultaneously under identical conditions

  • Use the same samples, concentrations, and detection methods

  • Compare staining patterns, band positions, and signal intensities

• Epitope mapping analysis:

  • Determine which regions of GTF3C6 each antibody recognizes

  • Compare antibodies targeting different epitopes (e.g., N-terminal vs. C-terminal)

  • Consider how epitope location might affect detection in different applications

• Application-specific validation:

  For Western blot:

  • Compare band patterns and molecular weights (expect 35-38 kDa)

  • Test detection limit by serial dilution of lysate

  • Evaluate specificity using knockout/knockdown controls

  For IHC:

  • Compare staining patterns in the same tissue sections

  • Evaluate background levels and signal-to-noise ratio

  • Assess concordance with known expression patterns

• Cross-validation with orthogonal methods:

  • Correlate antibody-based detection with mRNA expression data

  • Compare results with epitope-tagged GTF3C6 detected via tag antibodies

  • Validate localization findings with fluorescent protein fusions

• Quantitative comparison metrics:

  • Signal-to-noise ratio calculation

  • Limit of detection determination

  • Coefficient of variation across replicates

  • Dynamic range assessment

  • Concordance analysis between antibodies (e.g., Pearson correlation)

• Documentation and reporting:

  • Record detailed metadata for each antibody (catalog number, lot, dilution)

  • Document optimization parameters for each application

  • Report comparative findings with appropriate statistical analysis

  • Include representative images showing comparison results

• Specialized validation for specific applications:

  • For IP: Compare pull-down efficiency by quantifying percent of input recovered

  • For IHC: Use automated scoring systems to objectively compare staining

  • For multiplex applications: Assess antibody compatibility in combination

This systematic approach provides objective data on antibody performance, enabling informed selection for specific research applications and enhancing result reliability.

How can I optimize GTF3C6 antibody staining for fluorescence microscopy?

Optimizing GTF3C6 antibody staining for fluorescence microscopy requires attention to several methodological details:

• Fixation method optimization:

  • Compare paraformaldehyde (2-4%) versus methanol fixation

  • Test dual fixation methods if necessary (brief PFA followed by methanol)

  • Optimize fixation time (typically 10-20 minutes at room temperature)

• Permeabilization protocol:

  • Since GTF3C6 is primarily nuclear, ensure adequate nuclear permeabilization

  • Test Triton X-100 (0.1-0.5%) versus saponin (0.1-0.3%)

  • Optimize permeabilization time (typically 5-15 minutes)

• Antigen retrieval considerations:

  • For fixed cells/tissues, adapt IHC retrieval methods

  • Test heat-induced epitope retrieval with TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 (alternative)

  • For cultured cells, mild retrieval may improve nuclear protein detection

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for nuclear proteins

  • Extend blocking time (1-2 hours) to reduce background

• Antibody concentration optimization:

  • Start with dilutions similar to IHC (1:500-1:2000)

  • Perform dilution series to identify optimal concentration

  • Consider longer incubation times (overnight at 4°C) at higher dilutions

• Secondary antibody selection:

  • Choose bright fluorophores (Alexa Fluor 488, 555, 647)

  • Select secondary antibodies with minimal cross-reactivity

  • Consider signal amplification systems for low abundance targets

• Counterstaining optimization:

  • Use DAPI or Hoechst for nuclear counterstaining

  • Consider additional markers for subcellular structures

  • Optimize mounting media (antifade properties)

• Technical considerations:

  • Include no-primary-antibody control

  • Use isotype control at same concentration

  • Perform single-color controls before attempting multiplexing

• Imaging parameters:

  • Optimize exposure settings to prevent saturation

  • Use the same acquisition parameters for comparative analyses

  • Consider deconvolution or super-resolution techniques for detailed localization

• Validation of staining pattern:

  • Compare localization with published data

  • Confirm nuclear localization with nuclear markers

  • Verify specificity using knockdown controls

Following these optimization steps will help achieve specific, reproducible staining of GTF3C6 for fluorescence microscopy applications.

What are the key considerations for using GTF3C6 antibodies in multicolor flow cytometry?

When incorporating GTF3C6 antibodies into multicolor flow cytometry panels, consider these methodological aspects:

• Antibody format selection:

  • Use directly conjugated antibodies when available (FITC-conjugated GTF3C6 antibodies are available)

  • For unconjugated antibodies, select secondary antibodies with minimal spectral overlap

  • Consider tandem dyes for expanded panel design

• Panel design considerations:

  • Assign GTF3C6 to a channel with appropriate sensitivity based on expected expression level

  • Balance fluorophore brightness with expected antigen density

  • Minimize spectral overlap with other markers in your panel

• Sample preparation optimization:

  • For nuclear protein detection, use robust fixation (2-4% paraformaldehyde)

  • Ensure complete permeabilization (0.1-0.5% Triton X-100 or commercially available permeabilization buffers)

  • Include protein transport inhibitors if measuring alongside cytokines

• Staining protocol considerations:

  • For intracellular staining, perform surface marker staining before fixation/permeabilization

  • Optimize antibody concentration through titration

  • Consider longer incubation times for intracellular targets (30-60 minutes)

  • Include washing steps with saponin-containing buffer to maintain permeabilization

• Critical controls:

  • Unstained cells

  • Fluorescence minus one (FMO) controls

  • Isotype controls at matching concentrations

  • Biological controls (positive and negative cell types)

  • Compensation controls for each fluorochrome

• Instrument setup and quality control:

  • Perform daily quality control with tracking beads

  • Set PMT voltages for optimal resolution

  • Adjust compensation using single-color controls

  • Consider application-specific instrument settings

• Analysis considerations:

  • Use appropriate gating strategies to identify cell populations

  • Consider comparing median fluorescence intensity (MFI) rather than percent positive

  • Use visualization tools (t-SNE, UMAP) for high-dimensional analysis

  • Account for autofluorescence in certain cell types

• Validation approach:

  • Confirm flow cytometry results with alternate methods

  • Compare expression patterns with Western blot data

  • Validate with cells having known GTF3C6 expression levels

• Troubleshooting strategies:

  • For weak signals, try alternative fixation/permeabilization protocols

  • Consider signal amplification systems for low abundance targets

  • Test alternative fluorophores if sensitivity is inadequate

These considerations will help ensure successful incorporation of GTF3C6 antibodies into multicolor flow cytometry applications.

How should I interpret unexpected GTF3C6 staining patterns in immunohistochemistry?

When encountering unexpected GTF3C6 staining patterns in immunohistochemistry, consider these interpretation guidelines and troubleshooting approaches:

• Pattern variations and their potential meanings:

  • Cytoplasmic instead of nuclear staining: May indicate protein mislocalization in certain disease states, trafficking issues, or antibody cross-reactivity

  • Heterogeneous expression within a tissue: Could reflect biological heterogeneity, cell cycle-dependent expression, or microenvironmental influences

  • Unexpected cell type specificity: May reveal previously unknown expression patterns or non-specific binding

• Technical versus biological variability assessment:

  • Replicate staining with the same antibody lot

  • Test alternative GTF3C6 antibodies recognizing different epitopes

  • Compare with validated positive control tissues (human urothelial carcinoma, human lung cancer, mouse lung tissue)

  • Test multiple samples of the same tissue type to determine pattern consistency

• Verification strategies:

  • Perform antibody validation controls (antigen pre-absorption, isotype controls)

  • Correlate with mRNA expression by in situ hybridization

  • Compare with GTF3C6 expression in public databases

  • Use genetic approaches (RNA interference) to confirm specificity

• Confounding factors to consider:

  • Fixation artifacts: Overfixation or delayed fixation can alter staining patterns

  • Retrieval efficiency: Different antigen retrieval methods may reveal different epitopes (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Post-translational modifications: May mask epitopes in certain cellular contexts

  • Protein-protein interactions: May obstruct antibody accessibility to epitopes

• Context-dependent interpretation:

  • Disease context: Altered localization may represent pathological changes

  • Developmental context: Expression patterns may differ during development

  • Treatment effects: Therapeutic interventions may alter expression or localization

• Documentation and reporting recommendations:

  • Document all unexpected patterns with representative images

  • Report antibody details, retrieval methods, and controls used

  • Describe pattern quantitatively (percentage of cells, staining intensity)

  • Compare with expected patterns based on literature

• Advanced verification approaches:

  • Laser capture microdissection followed by Western blot or mass spectrometry

  • Correlative light and electron microscopy for precise localization

  • Single-cell analysis to characterize heterogeneous populations

Unexpected staining patterns, when properly validated, may represent novel biological insights rather than technical artifacts.

What is the current state of research on GTF3C6's role in human disease?

While the search results do not provide comprehensive information on GTF3C6's role in specific diseases, we can infer current research directions from the available data and context:

• Cancer research applications:

  • GTF3C6 antibodies have been validated in human cancer tissues, including urothelial carcinoma and lung cancer

  • These validation studies suggest active investigation of GTF3C6's potential role in cancer biology

  • As a component of the RNA polymerase III transcription machinery, GTF3C6 may influence cancer cell growth through regulation of tRNA and other small RNA synthesis

• Potential disease mechanisms:

  • Altered RNA polymerase III activity has been linked to cancer progression through increased protein synthesis capacity

  • Dysregulation of GTF3C6 could potentially impact cellular growth regulation pathways

  • As a nuclear transcription factor component, GTF3C6 may have roles in disease-specific gene expression programs

• Methodological approaches in disease research:

  • Immunohistochemical analysis of GTF3C6 expression in patient samples

  • Correlation of expression levels with clinical parameters

  • Functional studies using cell line models of disease

• Research challenges and considerations:

  • Limited public data on GTF3C6 disease associations

  • Need for large-scale expression studies across disease states

  • Importance of validating antibody specificity in disease tissues

  • Requirement for functional studies to establish causality rather than correlation

• Emerging research directions:

  • Integration of GTF3C6 in multi-omics disease studies

  • Investigation of GTF3C6 as a potential biomarker

  • Exploration of regulatory mechanisms controlling GTF3C6 expression

  • Analysis of post-translational modifications in disease contexts

• Research tools development:

  • Continued refinement of antibodies for disease research applications

  • Development of tissue-specific knockout models

  • Creation of systems biology approaches to understand GTF3C6 in disease networks

Researchers investigating GTF3C6's role in disease should focus on validating expression changes with multiple methodologies and establishing functional consequences through mechanistic studies.

How can I integrate GTF3C6 antibody data with genomic and transcriptomic information?

Integrating GTF3C6 antibody-derived protein data with genomic and transcriptomic information provides a comprehensive understanding of GTF3C6 biology. Here's how to approach this integration:

• Multi-omics correlation approaches:

  • Compare protein levels (Western blot, IHC) with mRNA expression (RNA-seq, qPCR)

  • Correlate GTF3C6 protein levels with gene expression profiles to identify regulated pathways

  • Integrate with ChIP-seq data (if available) to connect binding sites with expression changes

  • Use public databases like All of Us Research Program's All by All tables for large-scale genomic associations

• Methodological integration strategies:

  • Sample matching: Use the same samples for proteomics and genomics when possible

  • Temporal analysis: Track changes across time points in both datasets

  • Cell type-specific analysis: Sort cells before analysis or use single-cell approaches

  • Pathway-level integration: Map both protein and transcript data to common pathways

• Bioinformatic approaches:

  • Use correlation networks to identify genes with similar expression patterns

  • Apply machine learning to integrate multiple data types

  • Perform enrichment analysis to identify biological processes associated with GTF3C6

  • Utilize Bayesian networks to infer causal relationships

• Validation strategies:

  • Confirm key findings with orthogonal methods

  • Use genetic perturbation (CRISPR, RNAi) to validate functional relationships

  • Perform rescue experiments to confirm specificity of observed effects

• Database resources for integration:

  • The All of Us Research Program provides extensive genomic and phenotypic data that can be correlated with antibody-derived protein data

  • Public repositories like GEO and SRA for transcriptomic data

  • The Cancer Genome Atlas (TCGA) for cancer-specific multi-omics data

  • GTEx for tissue-specific expression patterns

• Advanced integration techniques:

  • Spatial transcriptomics combined with immunohistochemistry

  • Single-cell multi-omics to correlate protein and RNA at the cellular level

  • Trajectory analysis to study GTF3C6 dynamics during cellular processes

  • Network analysis to place GTF3C6 in the context of larger regulatory systems

• Visualization and reporting:

  • Create integrated visualizations showing multiple data types

  • Report correlation statistics between protein and transcript levels

  • Provide biological context for observed relationships

  • Document methodological details for reproducibility

This integrated approach provides deeper insights than any single data type and helps place GTF3C6 within broader biological contexts.