SUPT16H Antibody, FITC conjugated

What is SUPT16H and why is it significant in chromatin research?

SUPT16H (Suppressor of Ty 16 Homolog) is a critical subunit of the FACT (FAcilitates Chromatin Transcription) complex, which functions as a histone chaperone involved in nucleosome reorganization. The FACT complex participates in multiple DNA-template processes including mRNA elongation, DNA replication, and DNA repair. During transcription elongation, FACT acts by both destabilizing and restoring nucleosomal structure, facilitating RNA polymerase II passage by promoting the dissociation of one histone H2A-H2B dimer from the nucleosome and subsequently reestablishing the nucleosome after polymerase passage . This dual functionality makes SUPT16H a valuable target for studying chromatin dynamics and transcriptional regulation.

What are the structural domains of SUPT16H and their functions?

SUPT16H consists of four distinct domains with specialized functions:

• N-terminal domain (NTD): Involved in protein-protein interactions

• Dimerization domain (DD): Mediates interaction with other proteins including SSRP1

• Middle domain (MD): Contains the lysine 674 (K674) acetylation site, which is critical for interactions with bromodomain proteins like BRD4

• C-terminal domain (CTD): Involved in histone binding and chaperone function

The middle domain is particularly significant as it undergoes post-translational modifications like acetylation that regulate SUPT16H function and interactions with other chromatin-associated proteins .

What applications are FITC-conjugated SUPT16H antibodies best suited for?

• Immunofluorescence microscopy to visualize SUPT16H localization

• Flow cytometry to quantify SUPT16H expression levels

• Chromatin immunoprecipitation followed by microscopy (ChIP-imaging)

• Live-cell imaging when using membrane-permeable variants

When selecting applications beyond ELISA, validation experiments are essential to confirm specificity and performance in your experimental system .

How should I determine the optimal dilution for SUPT16H FITC-conjugated antibody in different applications?

For optimal dilution determination:

• Begin with manufacturer-recommended dilutions (typically in the range of 1:100 to 1:500 for ELISA)

• Perform a titration experiment with serial dilutions (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

• Include both positive controls (cells/tissues known to express SUPT16H) and negative controls (knockdown cells or isotype controls)

• Analyze signal-to-noise ratio at each dilution to identify the concentration that provides maximum specific signal with minimal background

• Validate findings across multiple experimental replicates before proceeding with main experiments

This methodical approach ensures reliable and reproducible results while conserving valuable antibody resources .

What controls are essential when using SUPT16H antibodies in research applications?

Essential controls include:

• Positive tissue/cell controls: Cell lines with verified SUPT16H expression (e.g., HEK293T, HeLa, Jurkat, or NK-92 cells, all of which express detectable levels of acetylated SUPT16H)

• Negative controls:

  • Isotype control: IgG from the same species (rabbit) at matching concentration

  • SUPT16H-knockdown cells (siRNA or shRNA treated)

• Blocking peptide controls: Pre-incubation with immunizing peptide should abolish specific signal

• Cross-reactivity controls: Testing in tissues from different species if working across species boundaries

• Method controls: Secondary antibody-only controls to assess non-specific binding

How can I verify SUPT16H antibody specificity in my experimental system?

To verify antibody specificity:

• Western blotting: Confirm a single band at the expected molecular weight (140 kDa for full-length SUPT16H)

• Immunoprecipitation followed by mass spectrometry: Identify SUPT16H and known interacting partners (SSRP1, BRD4, TIP60)

• siRNA/shRNA knockdown: Demonstrate reduction in signal proportional to knockdown efficiency

• Competitive binding assay: Pre-incubation with immunizing peptide should eliminate specific binding

• Parallel testing with alternative antibodies targeting different epitopes of SUPT16H

• Expression system validation: Test in cells overexpressing tagged SUPT16H to confirm co-localization

How can SUPT16H antibodies be utilized to study its role in interferon signaling pathways?

To investigate SUPT16H's role in interferon signaling:

• Combine SUPT16H immunoprecipitation with proteomic analysis to identify interactions with interferon regulatory factors (IRFs)

• Use ChIP-seq with SUPT16H antibodies before and after interferon stimulation to map genome-wide binding changes

• Perform dual immunofluorescence with SUPT16H and interferon-stimulated gene (ISG) products to assess co-localization

• Utilize SUPT16H antibodies in proximity ligation assays (PLA) to detect direct interactions with components of interferon signaling pathways

• Compare histone modification patterns (H3K9me3, H3K27me3, H3ac) at ISG promoters in control versus SUPT16H-depleted cells

Research has demonstrated that SUPT16H genetic knockdown or pharmacological inhibition induces interferon and interferon-stimulated genes, suggesting a previously unappreciated role in regulating interferon responses .

What methodologies can be employed to study SUPT16H acetylation using antibodies?

To study SUPT16H acetylation:

• Immunoprecipitation with anti-acetyl lysine antibodies followed by SUPT16H detection (or vice versa)

• Generation or procurement of acetylation-specific antibodies targeting K674

• Treatment with deacetylase inhibitors to enhance acetylation signal

• Mass spectrometry analysis of immunoprecipitated SUPT16H to map acetylation sites

• Mutagenesis studies (K674R) combined with antibody detection to confirm specificity

• ChIP assays to determine how acetylation affects chromatin binding

Research has identified that SUPT16H is acetylated at lysine 674 in its middle domain by the TIP60 histone acetyltransferase, and this modification promotes interaction with BRD4 .

How can SUPT16H antibodies contribute to antiviral research applications?

For antiviral research applications:

• Use SUPT16H antibodies to monitor protein levels and localization during viral infection

• Perform ChIP-seq to map SUPT16H binding near interferon and antiviral gene loci

• Combine with viral protein immunostaining to assess co-localization during infection

• Study changes in SUPT16H post-translational modifications in response to viral challenge

• Monitor SUPT16H-BRD4 interactions during infection using co-immunoprecipitation

• Assess SUPT16H recruitment to viral replication centers

Studies have shown that inhibition of SUPT16H using curaxin 137 (CBL0137) induces interferon signaling and effectively inhibits infection by multiple viruses, including Zika, influenza, and SARS-CoV-2 .

What are the common technical challenges when working with FITC-conjugated SUPT16H antibodies?

Common technical challenges include:

• Photobleaching: FITC is susceptible to rapid photobleaching during fluorescence microscopy

  • Solution: Use anti-fade mounting media, minimize exposure time, consider alternative more photostable fluorophores for long-term imaging

• Autofluorescence: Cellular components may produce background in the FITC channel

  • Solution: Include proper controls, use spectral unmixing, consider longer wavelength conjugates

• Signal amplification limitations: Direct FITC conjugation may provide insufficient signal for low-abundance targets

  • Solution: Consider tyramide signal amplification (TSA) or other signal enhancement methods

• pH sensitivity: FITC fluorescence is optimal at pH 8.0 and decreases at lower pH

  • Solution: Ensure buffers are properly formulated and pH-controlled

How can I optimize fixation protocols when using SUPT16H antibodies for immunofluorescence?

For optimized fixation:

• Compare multiple fixation methods:

  • 4% paraformaldehyde (10-15 minutes at room temperature) - preserves structure but may mask some epitopes

  • Methanol (-20°C for 10 minutes) - better for nuclear proteins but can distort membranes

  • Combined protocols (brief PFA followed by methanol) - may provide better epitope accessibility

• Test antigen retrieval methods if signal is weak:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0)

  • Enzymatic retrieval (proteinase K, trypsin)

• Optimize permeabilization:

  • Titrate detergent concentration (0.1-0.5% Triton X-100 or 0.05-0.2% Saponin)

  • Adjust incubation time (5-30 minutes)

• Consider acetylation status:

  • If studying acetylated SUPT16H, include deacetylase inhibitors in buffers

What troubleshooting approaches should be considered when detecting SUPT16H-protein interactions?

For troubleshooting protein interaction studies:

• Cross-linking optimization:

  • Test different cross-linkers (formaldehyde, DSS, DSP) at varying concentrations

  • Adjust cross-linking time (5-30 minutes) to capture transient interactions

• Buffer composition adjustments:

  • Modify salt concentration (150-500mM NaCl)

  • Test different detergents (NP-40, Triton X-100, CHAPS)

  • Include specific inhibitors (phosphatase, protease, deacetylase) to preserve modifications

• Nuclear extraction techniques:

  • Compare different extraction methods to maintain nuclear protein complexes

  • Consider sonication vs. enzymatic digestion for chromatin-bound complexes

• Confirming specificity:

  • Use reciprocal IP approaches (as performed in SUPT16H-BRD4 interaction studies)

  • Include competing peptides as negative controls

How can SUPT16H antibodies be applied in single-cell research techniques?

For single-cell applications:

• Flow cytometry:

  • Use FITC-conjugated SUPT16H antibodies for quantitative analysis of expression levels across cell populations

  • Combine with cell cycle markers to assess cell cycle-dependent regulation

• Single-cell imaging:

  • Apply in high-content imaging platforms for population-level analysis of SUPT16H localization

  • Implement in live-cell imaging with cell-permeable antibody formats

• Single-cell ChIP:

  • Adapt antibodies for CUT&Tag or CUT&RUN protocols for single-cell epigenomic profiling

  • Combine with DNA FISH to correlate SUPT16H binding with specific genomic loci

• Mass cytometry (CyTOF):

  • Metal-conjugated SUPT16H antibodies can be integrated into multi-parameter single-cell analysis panels

What insights can SUPT16H antibodies provide about chromatin remodeling during cellular differentiation?

For studying differentiation processes:

• ChIP-seq time course:

  • Map SUPT16H occupancy changes during differentiation trajectories

  • Correlate with changes in histone modifications and transcriptional activity

• Interaction dynamics:

  • Track SUPT16H-BRD4 interactions during lineage commitment

  • Assess changes in SUPT16H association with different chromatin modifiers

• Post-translational modifications:

  • Monitor acetylation status of SUPT16H during differentiation

  • Correlate with functional outcomes in lineage specification

• Functional assessment:

  • Compare effects of SUPT16H inhibition at different differentiation stages

  • Determine cell type-specific dependencies on SUPT16H function

How does SUPT16H contribute to the regulation of specific gene expression programs?

To investigate gene-specific regulation:

• Target gene analysis:

  • Perform ChIP-qPCR at specific promoters before and after SUPT16H manipulation

  • Correlate SUPT16H binding with histone modification changes (H3K9me3, H3K27me3, H3ac)

• Genomic context assessment:

  • Compare SUPT16H binding patterns at active versus repressed genes

  • Analyze SUPT16H recruitment to different genomic features (promoters, enhancers, gene bodies)

• Functional domains:

  • Use domain-specific antibodies to determine which SUPT16H domains associate with specific gene targets

  • Create domain deletion mutants to assess functional contributions

• Co-regulator dependencies:

  • Determine if SUPT16H-mediated gene regulation requires BRD4, TIP60, or other partners

  • Test combinatorial factors across different gene sets

Research has demonstrated that SUPT16H contributes to the silencing of specific genes, including HIV-1 proviruses and interferon-stimulated genes, through associations with epigenetic silencing enzymes like EZH2 and HDAC1 .

How should researchers interpret conflicting results between different SUPT16H detection methods?

When facing conflicting results:

• Consider epitope accessibility:

  • Different antibodies target different regions (internal region, C-terminus, specific domains)

  • Some epitopes may be masked in certain contexts or interactions

• Post-translational modifications:

  • Acetylation at K674 may affect antibody recognition

  • Other modifications may influence protein detection

• Context-dependent associations:

  • SUPT16H has different binding partners in different cellular processes

  • Protein complex formation may shield epitopes

• Methodological limitations:

  • Create a comparison table of detection methods listing strengths and limitations

  • Consider orthogonal approaches for validation

What quantitative methods are recommended for analyzing SUPT16H occupancy on chromatin?

For quantitative analysis:

• ChIP-seq normalization approaches:

  • Use spike-in controls (e.g., Drosophila chromatin)

  • Apply appropriate normalization methods (RPKM, TMM)

• Peak calling considerations:

  • Select appropriate algorithms based on expected binding profiles

  • Consider broader domains versus sharp peaks

• Comparative metrics:

  • Calculate occupancy changes using log2 fold change

  • Apply statistical tests appropriate for ChIP data (DESeq2, edgeR)

• Correlation analysis:

  • Compare SUPT16H binding with histone modifications

  • Assess co-occupancy with transcription factors

• Visualization approaches:

  • Generate heatmaps of SUPT16H binding across gene categories

  • Create metaplots centered on transcription start sites

How can researchers distinguish between direct and indirect effects of SUPT16H manipulation in cellular systems?

To differentiate direct from indirect effects:

• Temporal analysis:

  • Perform time-course experiments after SUPT16H perturbation

  • Early changes (0-4 hours) are more likely direct consequences

• Rapid protein depletion:

  • Use degron-based systems for acute protein loss rather than siRNA

  • Compare acute versus chronic depletion phenotypes

• Rescue experiments:

  • Reintroduce wild-type versus mutant SUPT16H (K674R, domain deletions)

  • Assess which phenotypes are rescued

• Direct binding assessment:

  • Correlate ChIP-seq data with gene expression changes

  • Genes with SUPT16H occupancy and expression changes are likely direct targets

• Mechanistic dissection:

  • Use inhibitors of downstream pathways to block indirect effects

  • Perform epistasis experiments with known interactors

How does SUPT16H function compare with its binding partner SSRP1 within the FACT complex?

Comparative analysis of FACT components:

While SUPT16H has been shown to undergo acetylation that regulates its interactions, such modifications of SSRP1 are less characterized. Additionally, SUPT16H appears to have SSRP1-independent functions in gene silencing through associations with epigenetic modifiers .

What are the key differences between various commercially available SUPT16H antibodies?

Comparison of SUPT16H antibodies based on available data:

Selection should be based on the specific experimental requirements, target region of interest, and desired applications .

How do inhibitors of SUPT16H compare with genetic knockdown approaches in research applications?

Comparison of SUPT16H manipulation strategies:

Both pharmacological inhibition with CBL0137 and genetic knockdown via RNAi have been shown to induce interferon signaling, though CBL0137 appears to produce stronger effects, particularly relevant to antiviral applications .

What emerging technologies might enhance SUPT16H research using antibody-based approaches?

Emerging technologies include:

• Proximity-dependent labeling:

  • BioID or TurboID fusion proteins to identify context-specific SUPT16H interactomes

  • APEX2-based approaches for subcellular mapping of SUPT16H complexes

• Single-molecule imaging:

  • Super-resolution microscopy with specifically designed antibody fragments

  • Single-particle tracking to monitor SUPT16H dynamics at individual nucleosomes

• Combinatorial epigenomic profiling:

  • Multi-omics approaches combining SUPT16H ChIP-seq with RNA-seq, ATAC-seq

  • Simultaneous profiling of multiple chromatin features with SUPT16H

• Structural analysis:

  • Cryo-EM of SUPT16H complexes using antibodies for complex stabilization

  • Hydrogen-deuterium exchange mass spectrometry with antibody epitope mapping

How might SUPT16H research contribute to understanding treatment resistance in cancer?

Future research directions in cancer:

• Therapeutic resistance mechanisms:

  • Investigate SUPT16H-mediated chromatin changes in drug-resistant cancer cells

  • Target SUPT16H pathways to overcome resistance to epigenetic therapies

• Biomarker development:

  • Assess SUPT16H acetylation status as a predictive biomarker for BRD4 inhibitor response

  • Develop immunohistochemistry protocols for clinical samples

• Combination therapy approaches:

  • Test CBL0137 in combination with immune checkpoint inhibitors

  • Explore synergies between SUPT16H inhibition and conventional chemotherapies

• Cancer immunotherapy connections:

  • Further investigate the role of SUPT16H in interferon signaling within the tumor microenvironment

  • Explore how SUPT16H manipulation might enhance immunotherapy responses

What are the implications of SUPT16H research for understanding infectious disease mechanisms?

Infectious disease research implications:

• Broad-spectrum antiviral development:

  • Further characterize how SUPT16H inhibition affects diverse viral families

  • Optimize CBL0137 derivatives for antiviral applications

• Host-directed therapy approaches:

  • Target SUPT16H pathways as alternative to direct-acting antivirals

  • Develop combination approaches targeting both viral and host factors

• Immune modulation strategies:

  • Harness SUPT16H-mediated interferon induction for vaccine adjuvant development

  • Explore effects on natural killer cell function in various infection models

• Resistance mechanism investigation:

  • Study how viruses might evolve to counter SUPT16H-mediated immune activation

  • Identify viral proteins that directly interact with SUPT16H