SUPT16H Antibody

Basic Research Questions

• What is SUPT16H and what is its biological function?

  SUPT16H (Suppressor of Ty 16 Homolog) is a component of the FACT (Facilitates Chromatin Transcription) complex, which functions as a general chromatin factor that reorganizes nucleosomes. The FACT complex, including SUPT16H, is involved in multiple DNA-dependent processes including mRNA elongation, DNA replication, and DNA repair .

  During transcription elongation, the FACT complex acts as a histone chaperone that both destabilizes and restores nucleosomal structure. It facilitates RNA polymerase II passage by promoting the dissociation of one histone H2A-H2B dimer from the nucleosome, then subsequently promotes nucleosome reestablishment following polymerase passage . SUPT16H is also involved in the phosphorylation of p53/TP53 at 'Ser-392' via its association with casein kinase II (CK2) and participates in vitamin D-coupled transcription regulation through its association with the WINAC complex .

• What are the common applications for SUPT16H antibodies in research?

  SUPT16H antibodies are utilized in multiple experimental applications:

  Application	Common Usage	Typical Dilutions
  Western Blotting (WB)	Protein detection and quantification	1:500-1:50000
  Immunohistochemistry (IHC)	Tissue localization	1:20-1:2000
  Immunofluorescence (IF/ICC)	Cellular localization	1:50-1:2000
  Immunoprecipitation (IP)	Protein complex isolation	0.5-4.0 μg for 1-3 mg lysate
  ELISA	Quantitative protein detection	1:10000
  ChIP-Seq	Chromatin binding analysis	Application-specific

  The optimal application depends on your specific research question. For chromatin-related studies, ChIP-Seq and IP are particularly valuable, while cellular localization studies benefit from IF/ICC approaches .

• What is the expected molecular weight of SUPT16H in Western blot analysis?

  When performing Western blot analysis of SUPT16H:

  • The calculated molecular weight is approximately 120 kDa based on amino acid sequence

  • The observed molecular weight is typically 135-140 kDa in most cell and tissue lysates

  This discrepancy between calculated and observed molecular weights is likely due to post-translational modifications of the protein. When validating a new SUPT16H antibody, expect to see a band at approximately 140 kDa in Western blots. Different tissue samples and cell lines consistently show the 135-140 kDa band, including LNCaP, MCF-7, HeLa, HEK-293, HepG2, Jurkat, HSC-T6, NIH/3T3, and RAW 164.7 cells .

• What species reactivity should be considered when selecting a SUPT16H antibody?

  SUPT16H antibody reactivity varies significantly between commercial sources. When selecting an appropriate antibody, consider these common reactivity patterns:

  Species	Availability	Notes
  Human	Most common	Validated in multiple cell lines and tissues
  Mouse	Widely available	Tested in brain tissue and multiple cell lines
  Rat	Less common	Validated in specific tissue contexts
  Other mammals	Limited	Some antibodies cross-react with dog, pig, cow, rabbit, horse, monkey samples

  For cross-species studies, antibodies targeting highly conserved regions (particularly C-terminal domains) offer broader reactivity . Always validate the antibody in your specific species and experimental context before proceeding with full-scale experiments, especially when working with non-human/mouse/rat samples.

• What are the main types of SUPT16H antibodies available for research?

  SUPT16H antibodies are available in several formats with different characteristics:

  Type	Host	Advantages	Common Applications
  Polyclonal	Rabbit	Recognizes multiple epitopes, higher sensitivity	WB, IHC, IF/ICC
  Polyclonal	Goat	Alternative specificity profile	ELISA, WB
  Monoclonal	Mouse	Consistent lot-to-lot reproducibility, high specificity	WB, IHC, IF/ICC
  Recombinant Monoclonal	Rabbit	Superior reproducibility, defined specificity	WB

  Antibodies target different regions of SUPT16H:

  • N-terminal region antibodies (AA 77-126)

  • Internal region antibodies (AA 187-282, AA 608-715)

  • C-terminal region antibodies

  The epitope location can affect detection efficiency, especially if your protein of interest has undergone post-translational modifications or is part of a complex .

Advanced Research Questions

• How should I optimize SUPT16H antibody concentration for Western blotting to detect endogenous protein levels?

  Optimizing SUPT16H antibody concentration for Western blotting requires a systematic approach:

  1. Initial titration: Begin with a dilution range between 1:500-1:2000 for most polyclonal antibodies and 1:1000-1:5000 for monoclonal antibodies

  2. Sample preparation considerations:

     • Use RIPA or NP-40 based lysis buffers with protease inhibitors

     • Load 20-50 μg of total protein per lane

     • Include phosphatase inhibitors if studying phosphorylated forms

  3. Positive control selection: Include lysates from cells known to express SUPT16H at detectable levels (HeLa, MCF-7, or HEK-293 cells are recommended)

  4. Blocking optimization: 5% non-fat milk in TBST works well for most applications, but switch to 5% BSA if background is high

  5. Signal development: For low abundance detection, consider using ECL substrate with extended exposure times or higher sensitivity detection systems

  If detecting endogenous SUPT16H remains challenging, concentrate your samples using immunoprecipitation before Western blotting. The expected 135-140 kDa band should be clearly visible with minimal background when optimization is successful .

• What are the key considerations for using SUPT16H antibodies in ChIP-Seq experiments?

  When performing ChIP-Seq with SUPT16H antibodies, several critical factors must be addressed:

  1. Antibody selection: Use ChIP-validated antibodies specifically (such as those mentioned in publications using ChIP-Seq applications)

  2. Crosslinking optimization:

     • Use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking

     • For FACT complex components like SUPT16H, dual crosslinking with 1.5 mM EGS followed by formaldehyde can improve results

  3. Sonication parameters:

     • Optimize sonication to generate DNA fragments of 200-500 bp

     • Verify fragmentation efficiency by gel electrophoresis before proceeding

  4. Antibody amount:

     • Use 5-10 μg of antibody per ChIP reaction

     • Include appropriate IgG controls from the same species

  5. Validation approaches:

     • Verify enrichment at known SUPT16H binding sites by qPCR before sequencing

     • Include input controls and normalize appropriately during analysis

  6. Data analysis considerations:

     • SUPT16H typically shows broader peaks than transcription factors

     • Analyze correlation with active transcription markers (H3K4me3, H3K27ac)

     • Consider overlap with RNA Polymerase II binding sites

  SUPT16H ChIP-Seq experiments can provide valuable insights into the role of the FACT complex in chromatin remodeling during transcription, replication, and DNA repair processes .

• How can I validate the specificity of a SUPT16H antibody for my particular application?

  Rigorous validation of SUPT16H antibody specificity is essential for generating reliable data. Implement the following comprehensive validation strategy:

  1. Positive and negative controls:

     • Positive: Tissues/cells known to express SUPT16H (e.g., actively dividing cells like bone marrow and epithelial layers)

     • Negative: SUPT16H knockout or knockdown cells (using siRNA or CRISPR)

  2. Multiple antibody comparison:

     • Use antibodies targeting different epitopes of SUPT16H

     • Compare monoclonal vs. polyclonal antibodies from different sources

  3. Cross-reactivity assessment:

     • Test in multiple species if working with non-human models

     • Conduct peptide competition assays with the immunizing peptide

  4. Application-specific validation:

     • For Western blot: Verify correct molecular weight (135-140 kDa)

     • For IHC/IF: Include appropriate blocking peptides

     • For IP: Confirm pull-down by mass spectrometry

     • For ChIP: Validate enrichment at known targets by qPCR

  5. Orthogonal methods:

     • Correlate protein expression with mRNA levels

     • Verify localization patterns using tagged SUPT16H constructs

  Antibody specificity should be re-evaluated when changing experimental conditions, cell types, or when using new lots of the same antibody .

• What are the optimal tissue preparation methods for SUPT16H immunohistochemistry?

  Successful SUPT16H immunohistochemistry requires careful attention to tissue preservation and antigen retrieval:

  1. Fixation parameters:

     • Optimal fixation: 10% neutral buffered formalin for 24-48 hours

     • Avoid overfixation which can mask epitopes

  2. Antigen retrieval methods:

     • Primary recommendation: TE buffer (pH 9.0) heat-induced epitope retrieval

     • Alternative approach: Citrate buffer (pH 6.0) if TE buffer yields suboptimal results

  3. Section thickness:

     • Use 4-5 μm sections for optimal antibody penetration

  4. Blocking procedure:

     • Block with 5-10% normal serum from the same species as the secondary antibody

     • Include 0.1-0.3% Triton X-100 for improved penetration

  5. Antibody dilution and incubation:

     • Start with 1:100-1:300 dilutions for most SUPT16H antibodies

     • Incubate overnight at 4°C for optimal sensitivity

  6. Signal detection system:

     • Use polymer-based detection systems for enhanced sensitivity

     • Consider tyramide signal amplification for low-abundance detection

  7. Positive control tissues:

     • Human tonsillitis tissue and human spleen tissue show consistent SUPT16H expression

     • Colon tissue has also been validated for SUPT16H detection

  Always optimize each step for your specific tissue type and antibody combination to achieve maximum specificity with minimal background.

• How do post-translational modifications affect SUPT16H detection with antibodies?

  Post-translational modifications (PTMs) significantly impact SUPT16H detection and function analysis:

  1. Common SUPT16H modifications:

     • Phosphorylation: Multiple serine/threonine residues

     • Ubiquitination: Involved in protein turnover

     • SUMOylation: May affect chromatin association

  2. Impact on antibody detection:

     • Epitope masking: PTMs can block antibody access to recognition sites

     • Conformational changes: Modifications alter protein folding

     • Molecular weight shifts: Phosphorylation typically causes higher apparent MW (explaining the 120 kDa calculated vs. 135-140 kDa observed disparity)

  3. Modification-specific detection strategies:

     • Use phosphatase treatment to verify phosphorylation-dependent recognition

     • Employ modification-specific antibodies when studying particular PTMs

     • Consider domain-specific antibodies to target regions less affected by PTMs

  4. Functional significance:

     • SUPT16H phosphorylation status changes during the cell cycle

     • DNA damage induces specific modification patterns

     • The RNF40 ubiquitin ligase cooperates with SUPT16H during DNA double-strand break repair

  When studying SUPT16H in specific functional contexts (e.g., DNA repair), consider how relevant PTMs might affect antibody recognition and employ appropriate controls to account for modification-dependent detection variability.

• What are the best practices for using SUPT16H antibodies in co-immunoprecipitation to study protein-protein interactions?

  When performing co-immunoprecipitation (co-IP) with SUPT16H antibodies to study protein interactions:

  1. Lysis buffer optimization:

     • Use mild non-denaturing buffers (e.g., 20 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40)

     • Include protease and phosphatase inhibitors

     • Consider adding low concentrations of non-ionic detergents (0.1% NP-40) to reduce non-specific binding

  2. Antibody selection for IP:

     • Choose antibodies validated for immunoprecipitation (IP) applications

     • For mouse brain tissue, specific SUPT16H antibodies have been validated for IP

     • Consider using 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  3. Pre-clearing strategy:

     • Pre-clear lysates with protein A/G beads to remove non-specific binding proteins

     • Use matched IgG control from the same species as the SUPT16H antibody

  4. Co-IP validation approaches:

     • Reciprocal IP: Confirm interactions by IP with antibodies against suspected binding partners

     • Include known FACT complex components (SSRP1) as positive controls

     • Use stringent washing conditions to eliminate weak/non-specific interactions

  5. Expected interaction partners:

     • Primary: SSRP1 (other FACT complex component)

     • Secondary: Histones (especially H2A/H2B), RNA Polymerase II, CK2 (casein kinase II)

     • Context-specific: VDR and WINAC complex components during vitamin D-mediated transcription

  6. Detection method optimization:

     • Use 4-15% gradient gels for optimal separation of interaction partners

     • Consider silver staining or mass spectrometry for unbiased partner identification

  When studying SUPT16H interactions, remember that some interactions may be transient or context-dependent (e.g., during specific DNA damage responses or transcriptional events) .

• How does SUPT16H expression and localization vary across different cell types and tissues?

  SUPT16H expression and localization exhibit specific patterns across cells and tissues:

  1. Expression patterns:

     • Highest expression: Actively dividing cells, particularly in tissues with high rates of cell proliferation such as bone marrow and epithelial layers

     • Consistent detection: Validated in human tonsillitis tissue, human spleen tissue, and colon tissue

     • Cell lines: Well-expressed in LNCaP, MCF-7, HeLa, HEK-293, HepG2, Jurkat, HSC-T6, NIH/3T3, and RAW 164.7 cells

  2. Subcellular localization:

     • Primary localization: Nuclear, with predominant association with chromatin

     • Cell cycle-dependent variations: Redistribution during mitosis

     • Response to stimuli: Recruitment to sites of DNA damage

  3. Visualization methods:

     • Immunofluorescence optimal dilutions: 1:500-1:2000

     • Nuclear counterstaining: DAPI or Hoechst recommended

     • Z-stack imaging: Recommended for accurate nuclear localization assessment

  4. Methodological considerations:

     • Fixation: 4% paraformaldehyde (10-15 minutes) preserves nuclear architecture

     • Permeabilization: 0.1-0.3% Triton X-100 required for nuclear antibody access

     • Blocking: 3-5% BSA or normal serum reduces non-specific binding

  Understanding SUPT16H's expression and localization patterns is crucial for contextualizing its function in chromatin remodeling across different physiological and pathological conditions.

• What approaches can be used to study the dynamics of SUPT16H recruitment during DNA repair processes?

  To investigate SUPT16H dynamics during DNA repair:

  1. Induction of DNA damage:

     • Site-specific damage: Use laser microirradiation with live-cell imaging

     • Global damage: UV irradiation, ionizing radiation, or radiomimetic drugs

     • Chemical induction: Treatments with H₂O₂, hydroxyurea, or etoposide

  2. Temporal analysis approaches:

     • Live-cell imaging: Use fluorescently tagged SUPT16H constructs

     • Fixed-cell timecourse: Immunofluorescence at multiple timepoints post-damage

     • Biochemical fractionation: Track chromatin association over time

  3. Co-localization studies:

     • DNA damage markers: γH2AX, 53BP1, RAD51

     • Repair pathway components: Components of HR, NHEJ, or BER pathways

     • Chromatin modifiers: Histone variants, modification enzymes

  4. Functional analysis methods:

     • SUPT16H depletion: siRNA or CRISPR-based approaches

     • Domain mutations: Structure-function analysis of recruitment requirements

     • Inhibitor studies: Target interacting partners or upstream signaling

  5. Advanced techniques:

     • ChIP-Seq following damage: Map genome-wide SUPT16H redistribution

     • iPOND (isolation of Proteins On Nascent DNA): Study replication fork-associated repair

     • Proximity labeling: BioID or APEX2 fusions to identify transient interactions

  Research has shown that SUPT16H cooperates with the H2B ubiquitin ligase RNF40 during DNA double-strand break repair, inducing dynamic changes in chromatin structure . These approaches can help elucidate the mechanisms by which the FACT complex facilitates access to damaged DNA while maintaining genomic integrity.

• How can I effectively use SUPT16H antibodies in multiplexed immunofluorescence to study chromatin dynamics?

  For effective multiplexed immunofluorescence using SUPT16H antibodies:

  1. Antibody panel design:

     • Select primary antibodies from different host species (Rabbit anti-SUPT16H can be paired with mouse, goat, or rat antibodies targeting other proteins)

     • Include markers for:

       • FACT complex (SSRP1)

       • Chromatin states (histone modifications)

       • Transcriptional activity (RNA Pol II, transcription factors)

       • Cell cycle phase indicators (when relevant)

  2. Sequential staining protocols:

     • Apply antibodies sequentially if using same-species primaries

     • Consider tyramide signal amplification (TSA) for sequential same-species detection

     • Use appropriate controls for antibody stripping between rounds

  3. Imaging optimization:

     • Use spectral unmixing to resolve overlapping fluorophore emissions

     • Employ structured illumination or confocal microscopy for optimal resolution

     • Consider super-resolution techniques for detailed co-localization studies

  4. Analysis approaches:

     • Quantify co-localization using Pearson's or Mander's coefficients

     • Perform proximity analysis using distance measurements

     • Analyze chromatin texture changes in relation to SUPT16H distribution

  5. Validated applications:

     • SUPT16H antibodies have been successfully used in immunofluorescence at dilutions of 1:50-1:200 and 1:500-1:2000

     • HeLa cells have been validated as a cellular system for SUPT16H IF studies

  When optimizing your protocol, include appropriate controls (secondary-only, isotype controls) and consider the order of antibody application based on signal strength and antibody affinity.

• What are the key considerations when comparing SUPT16H antibody results across different experimental platforms?

  When comparing SUPT16H antibody results across platforms:

  1. Platform-specific epitope accessibility:

     • Fixed tissues (IHC): Crosslinking can mask certain epitopes

     • Denatured samples (WB): Linear epitopes are exposed, conformational epitopes lost

     • Native conformation (IP): Tertiary structure may block certain epitopes

     • Consider using antibodies targeting different regions (N-terminal, internal, C-terminal) when comparing across platforms

  2. Normalization strategies:

     • WB: Normalize to loading controls (β-actin, GAPDH)

     • IHC/IF: Use internal controls and standardized acquisition parameters

     • ChIP: Compare to input and IgG controls

  3. Cross-platform validation approaches:

     • Orthogonal methods: Correlate protein detection (antibody-based) with mRNA expression

     • Multiple antibodies: Use different clones targeting distinct epitopes

     • Genetic controls: Include knockdown/knockout samples across all platforms

  4. Quantification considerations:

     • Signal intensity range: Each method has different dynamic ranges

     • Background determination: Platform-specific background correction methods

     • Statistical analysis: Apply platform-appropriate statistical tests

  5. Documentation requirements:

     • Record complete antibody information: Catalog number, lot, dilution, incubation conditions

     • Include all experimental parameters that might affect results

     • Document image acquisition settings and analysis parameters

  Remember that discrepancies between platforms may reveal biologically relevant information about protein conformation, complex formation, or post-translational modifications rather than representing technical artifacts .

Technical Protocols

• What is the recommended protocol for SUPT16H antibody-based Western blotting?

  Detailed SUPT16H Western Blotting Protocol:

  1. Sample preparation:

     • Lyse cells in RIPA buffer with protease/phosphatase inhibitors

     • Determine protein concentration (BCA or Bradford assay)

     • Prepare samples with 4X Laemmli buffer + DTT, heat at 95°C for 5 minutes

  2. Gel electrophoresis:

     • Use 6-8% SDS-PAGE gels for optimal resolution of ~140 kDa SUPT16H

     • Load 20-50 μg total protein per lane

     • Include molecular weight marker and positive control (HeLa lysate recommended)

  3. Transfer parameters:

     • Transfer to PVDF membrane (0.45 μm pore size) for high MW proteins

     • Use wet transfer at 30V overnight at 4°C for large proteins

     • Verify transfer with reversible staining (Ponceau S)

  4. Blocking and antibody incubation:

     • Block with 5% non-fat milk in TBST for 1 hour at room temperature

     • Incubate with primary SUPT16H antibody at 1:500-1:2000 dilution overnight at 4°C

     • For higher sensitivity detection, use 1:5000-1:50000 dilution of mouse monoclonal antibody

     • Wash 3x with TBST, 5-10 minutes each

     • Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour

     • Wash 3x with TBST, 10 minutes each

  5. Detection and analysis:

     • Develop using ECL substrate

     • Expected band: 135-140 kDa

     • For quantification, normalize to housekeeping proteins

  This protocol has been optimized based on multiple validated SUPT16H antibody applications and should yield consistent results across different cell and tissue types.

• How can I troubleshoot common issues when working with SUPT16H antibodies?

  Comprehensive SUPT16H Antibody Troubleshooting Guide:

  1. No signal in Western blot:

     • Verify protein expression: SUPT16H is highly expressed in actively dividing cells

     • Increase protein loading: Try 50-100 μg total protein

     • Optimize antibody concentration: Try a more concentrated dilution (1:250-1:500)

     • Check transfer efficiency: Use stain-free gels or Ponceau S

     • Consider sample preparation: Use fresh lysates with complete protease inhibitors

     • Try different antibody: Use one targeting a different epitope

  2. Multiple bands or high background in Western blot:

     • Increase blocking stringency: 5% BSA instead of milk

     • Optimize antibody dilution: Use more dilute antibody solution

     • Increase wash duration and frequency

     • Use fresh/filtered antibody diluent

     • Check for sample degradation: Add additional protease inhibitors

  3. Weak or no signal in IHC/IF:

     • Optimize antigen retrieval: Try both pH 9.0 TE buffer and pH 6.0 citrate buffer

     • Increase antibody concentration: Use 1:20-1:50 dilution for weak signals

     • Extend primary antibody incubation time: Overnight at 4°C

     • Use signal amplification: Biotin-streptavidin or tyramide systems

     • Check fixation parameters: Overfixation can mask epitopes

  4. Non-specific staining in IHC/IF:

     • Optimize blocking: Use 3-5% serum from secondary antibody host species

     • Increase antibody dilution: Use 1:200-1:2000

     • Add 0.1-0.3% Triton X-100 for nuclear proteins

     • Perform peptide competition assay to confirm specificity

  5. Inefficient immunoprecipitation:

     • Increase antibody amount: Use 4-5 μg antibody per IP reaction

     • Optimize lysis conditions: Test different detergent concentrations

     • Extend incubation time: Overnight at 4°C with gentle rotation

     • Cross-link antibody to beads to prevent heavy chain interference

  For application-specific issues, refer to validation data from antibody manufacturers and published protocols using the specific SUPT16H antibody catalog numbers .