GTF2B Monoclonal Antibody

What is GTF2B and what role does it play in transcription?

GTF2B (General Transcription Factor IIB) functions as a critical component in transcription initiation by RNA polymerase II (Pol II). It participates in pre-initiation complex (PIC) formation and Pol II recruitment at promoter DNA. The protein forms a core initiation complex with TATA box-bound TBP (TATA-binding protein) and serves as a bridge between TBP and the Pol II-TFIIF complex. Following the onset of transcription, GTF2B is released from the PIC during the initiation-to-elongation transition and subsequently reassociates with TBP for the next transcription cycle. GTF2B also binds independently to two distinct DNA core promoter consensus sequence elements called IIB-recognition elements (BREs), which are located immediately upstream (BREu, 5'-GCGCGACGCC-3') and downstream (BREd, 5'-GATTGATG-3') of the TATA box .

Which applications are GTF2B monoclonal antibodies commonly used for?

GTF2B monoclonal antibodies are validated for multiple research applications, with varying effectiveness across different clones. Western blotting (WB) is the most universally supported application across available antibodies, with working dilutions typically between 1:500-1:5000 . Several antibodies also support ELISA applications, while some clones additionally support immunohistochemistry (IHC), immunocytochemistry (ICC), and immunoprecipitation (IP) . Researchers should select the appropriate clone based on their specific application requirements and experimental conditions.

What species reactivity can I expect from GTF2B monoclonal antibodies?

Species reactivity varies significantly between different GTF2B monoclonal antibody clones. The 2F6-A3 clone demonstrates cross-reactivity with human, mouse, and monkey samples . More broadly reactive antibodies like A10392 show reactivity against human, mouse, pig, rat, and dog samples . Some antibodies, such as PCRP-GTF2B-1H2, have been confirmed only for human reactivity . When working with non-human samples, researchers should carefully verify the cross-reactivity of their selected antibody before proceeding with experiments.

How should I optimize Western blot protocols for GTF2B detection?

For optimal Western blot detection of GTF2B (molecular weight approximately 34.8 kDa), follow these methodological considerations:

• Sample preparation: Due to nuclear localization of GTF2B, use nuclear extraction protocols with protease inhibitors to maximize yield.

• Loading control selection: For normalization, use nuclear protein controls such as Lamin B or Histone H3 rather than cytoplasmic controls like GAPDH.

• Blocking optimization: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute according to manufacturer recommendations (typically 1:500 for STJ99031 or 1:500-1:5000 for A10392) .

• Washing steps: Perform 4-5 washes with TBST, 5 minutes each.

• Secondary antibody selection: Use anti-mouse IgG secondary antibodies compatible with the isotype of your primary antibody (IgG2b for STJ99031 and A10392, or IgG2a for PCRP-GTF2B-1H2) .

• Detection method: Both chemiluminescence and fluorescence detection methods are compatible.

What controls should I include when using GTF2B antibodies in experimental work?

To ensure experimental rigor when working with GTF2B antibodies, implement the following controls:

• Positive control: Use cell lines with known GTF2B expression (most human cell lines express GTF2B, with HeLa and HEK293 cells being well-validated options).

• Negative control: Include a lane with recombinant GTF2B protein pre-incubated with the antibody to demonstrate specificity.

• Loading control: Include appropriate nuclear fraction markers.

• Isotype control: Use a non-specific mouse IgG of the same isotype as your GTF2B antibody.

• Knockdown/knockout validation: When possible, include GTF2B knockdown/knockout samples to confirm antibody specificity.

• Cross-reactivity assessment: If working with non-human samples, validate the antibody's reactivity in your species of interest against human positive controls .

How should GTF2B monoclonal antibodies be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody activity. Store GTF2B monoclonal antibodies at -20°C for long-term storage (up to one year from receipt) . For antibodies in solution form, avoid repeated freeze-thaw cycles by preparing single-use aliquots of no less than 20 μl before freezing . For short-term use (up to two weeks), antibodies can be stored at 4°C . Most commercial GTF2B antibodies are supplied in stabilizing buffers containing glycerol (typically 50%), which prevents freezing at -20°C and maintains antibody stability . Always check the expiration date and specific storage recommendations provided by the manufacturer for your particular antibody.

What are common troubleshooting strategies for weak or absent GTF2B signal in Western blots?

When encountering weak or absent GTF2B signals in Western blot applications, systematically address these potential issues:

• Sample preparation: Ensure proper nuclear extraction, as GTF2B is primarily nuclear.

• Protein degradation: Add fresh protease inhibitors to all buffers.

• Antibody dilution: Try a more concentrated antibody dilution (e.g., 1:250 instead of 1:500).

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C.

• Detection sensitivity: Use high-sensitivity ECL substrates or increase exposure time.

• Transfer efficiency: Verify protein transfer using reversible staining methods.

• Blocking optimization: Test alternative blocking agents (BSA vs. milk).

• Antibody quality: Check antibody expiration date and storage conditions.

• Species compatibility: Confirm the antibody's reactivity with your sample species.

• Post-translational modifications: Consider that phosphorylation or acetylation might affect epitope recognition .

How can I optimize immunoprecipitation protocols using GTF2B antibodies?

For successful immunoprecipitation of GTF2B complexes:

• Lysate preparation: Use gentle lysis buffers (e.g., 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) supplemented with protease and phosphatase inhibitors.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody selection: Use antibodies specifically validated for IP, such as PCRP-GTF2B-1H2 .

• Antibody amount: Use 2-5 μg of antibody per 500 μg of protein lysate.

• Incubation conditions: Incubate antibody-lysate mixture overnight at 4°C with gentle rotation.

• Bead selection: Use protein G beads for mouse IgG2a and protein A beads for mouse IgG2b antibodies.

• Washing stringency: Perform at least 4 washes with increasingly stringent buffers to reduce background.

• Elution strategy: Use either acidic elution (0.1 M glycine, pH 2.5) followed by immediate neutralization, or direct SDS boiling, depending on downstream applications.

• Control IPs: Always include an isotype control to identify non-specific interactions.

How can GTF2B antibodies be employed in ChIP experiments to study transcription factor binding?

Chromatin immunoprecipitation (ChIP) with GTF2B antibodies enables investigation of transcription initiation complexes at specific genomic loci. For optimal ChIP protocols:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature for protein-DNA crosslinking.

• Sonication: Optimize sonication conditions to generate 200-500 bp DNA fragments.

• Antibody selection: Choose GTF2B antibodies recognizing epitopes that remain accessible in crosslinked chromatin.

• Antibody amount: Use 3-5 μg of antibody per ChIP reaction.

• Preclearing: Reduce background by preclearing chromatin with protein A/G beads.

• Controls: Include input DNA, IgG control, and positive control (antibody against a known transcription factor).

• Washing: Use increasingly stringent wash buffers to reduce non-specific binding.

• Analysis methods: Analyze results using qPCR for targeted loci or sequencing (ChIP-seq) for genome-wide binding profiles.

• Data interpretation: Compare GTF2B binding patterns with other PIC components (TBP, Pol II) to identify active promoters.

• Validation: Validate findings with reporter assays or gene expression analysis.

How does GTF2B post-translational modification affect antibody recognition?

GTF2B undergoes several post-translational modifications that can impact antibody recognition, particularly acetylation. GTF2B can be acetylated at multiple sites, with autoacetylation at Lys-238 specifically known to stimulate transcription activation . These modifications may alter epitope accessibility or recognition by certain antibodies. When investigating modified forms of GTF2B:

• Antibody selection: Choose antibodies raised against epitopes distant from known modification sites.

• Modification-specific antibodies: For studies focusing on post-translational modifications, consider generating or sourcing modification-specific antibodies.

• Sample preparation: Preserve modifications by adding deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) to lysis buffers.

• Multiple antibody validation: Use different GTF2B antibodies recognizing distinct epitopes to confirm results.

• Mass spectrometry: Combine antibody-based detection with mass spectrometry to identify specific modification patterns.

What are the key differences between available GTF2B monoclonal antibody clones?

The table below provides a comparative analysis of three commercially available GTF2B monoclonal antibody clones:

When selecting between these clones, researchers should consider their specific application requirements, species of interest, and technical specifications such as isotype compatibility with secondary detection systems .

How should researchers validate a new GTF2B antibody before full experimental implementation?

Before implementing a new GTF2B antibody in critical experiments, conduct these validation steps:

• Western blot analysis: Confirm a single band at the expected molecular weight (34.8 kDa) in positive control samples.

• Positive controls: Test antibody performance in cell lines known to express GTF2B (e.g., HeLa, HEK293).

• Knockdown/knockout validation: Compare signal between wild-type and GTF2B-depleted samples.

• Cross-reactivity assessment: Test reactivity against closely related transcription factors.

• Application-specific validation: Perform pilot experiments for each intended application.

• Species cross-reactivity: Validate performance in each species of interest.

• Lot-to-lot consistency: When obtaining new lots, compare performance to previously validated lots.

• Signal-to-noise assessment: Evaluate background levels under different experimental conditions.

• Reproducibility: Ensure consistent results across multiple experiments.

• Literature comparison: Compare your findings with published results using the same or different antibodies.

How can GTF2B antibodies be used to study transcription dynamics in single cells?

Emerging single-cell applications for GTF2B antibodies include:

• Single-cell immunofluorescence: Use ICC-validated GTF2B antibodies (such as A10392) with high-resolution microscopy to visualize nuclear distribution patterns.

• Proximity ligation assays (PLA): Combine GTF2B antibodies with antibodies against other transcription factors to visualize protein-protein interactions at the single-cell level.

• Live-cell imaging: Create fluorescent protein fusions validated with antibody recognition to track GTF2B dynamics in real-time.

• Single-cell ChIP-seq: Adapt standard ChIP protocols using GTF2B antibodies to analyze chromatin binding in individual cells.

• Mass cytometry (CyTOF): Conjugate GTF2B antibodies with heavy metal isotopes for high-dimensional single-cell analysis.

• Spatial transcriptomics: Combine GTF2B immunostaining with in situ RNA detection to correlate transcription factor binding with gene expression at the single-cell level.

What considerations are important when using GTF2B antibodies in disease-specific research?

When applying GTF2B antibodies to disease-specific research contexts:

• Cell type specificity: Different cell types may express varying levels of GTF2B or tissue-specific isoforms.

• Disease-associated mutations: Mutations in GTF2B might affect epitope recognition by certain antibodies.

• Altered post-translational modifications: Disease states may induce changes in GTF2B acetylation patterns .

• Expression level changes: Calibrate antibody concentrations based on potential changes in GTF2B expression in disease states.

• Sample preparation: Disease tissues may require optimized extraction protocols to maintain protein integrity.

• Controls: Include both healthy and disease-specific positive controls.

• Background interference: Some pathological tissues may exhibit higher background staining, requiring additional blocking optimization.

• Cross-reactivity: Validate absence of cross-reactivity with disease-specific proteins that might be upregulated.

• Reproducibility: Disease heterogeneity may necessitate larger sample sizes to obtain reliable results.