SUPT16H Antibody, HRP conjugated

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

SUPT16H (Suppressor of Ty 16 Homolog) functions as a critical component of the FACT complex, forming a heterodimer with SSRP1 to facilitate chromatin transcription. This complex serves dual roles: as a histone chaperone promoting H2A-H2B dimer dissociation from nucleosomes, allowing RNA polymerase II access to chromatin templates, and as an initiation and elongation factor that colocalizes with RNA polymerase II to enable transcriptional activity . SUPT16H contains multiple functional domains including the N-terminal domain (NTD), dimerization domain (DD), middle domain (MD), and C-terminal domain (CTD), each contributing to its diverse biological activities . The protein's involvement in chromatin accessibility and transcriptional regulation makes it essential for understanding fundamental epigenetic mechanisms guiding development and disease processes.

What are the critical considerations when selecting a SUPT16H antibody for immunological techniques?

When selecting a SUPT16H antibody, researchers must consider several factors depending on their experimental objectives. First, evaluate the target specificity - whether the antibody recognizes internal regions, C-terminal domains, or specific amino acid sequences within SUPT16H. For instance, antibody ABIN190913 targets an internal peptide sequence DLTNKEGKKPEEK , while others target specific amino acid regions like AA 608-715 or AA 187-282 . Second, consider species cross-reactivity - some antibodies show reactivity across multiple species (human, mouse, cow, pig), while others are species-specific . Third, assess the validation status for your intended application - some antibodies are verified for multiple techniques (Western blot, immunohistochemistry, immunofluorescence, ELISA), while others have limited validation . Finally, examine the antibody type (polyclonal vs. monoclonal) and host species, as these factors influence specificity, batch consistency, and compatibility with other reagents in multiplexed experiments.

What optimal dilutions and sample preparations are recommended for SUPT16H antibody applications?

Optimal dilutions vary significantly by application type and specific antibody. For Western blotting applications, dilutions typically range from 1:500-1:2000 as recommended for antibody 20551-1-AP . For immunohistochemistry, more concentrated solutions (1:20-1:200) are generally required . For immunoprecipitation, quantitative guidelines suggest using 0.5-4.0 μg of antibody for every 1.0-3.0 mg of total protein lysate .

Sample preparation considerations include antigen retrieval methods - particularly for fixed tissues in immunohistochemistry applications. Proteintech's antibody validation data recommends TE buffer pH 9.0 for optimal antigen retrieval, with citrate buffer pH 6.0 as an alternative . For brain tissue samples, standard lysis buffers containing protease inhibitors have proven effective for both Western blot and immunoprecipitation applications, as demonstrated by positive detection in mouse brain tissue . Importantly, researchers should conduct titration experiments with their specific sample types, as optimal conditions can be sample-dependent.

How can researchers optimize SUPT16H detection in Western blotting experiments?

For optimal SUPT16H detection in Western blotting, researchers should consider several technical aspects. First, molecular weight expectations - SUPT16H has a calculated molecular weight of 120 kDa but typically appears at approximately 140 kDa on SDS-PAGE gels , requiring appropriate ladder selection and gel percentage optimization. Second, blocking conditions significantly impact background noise - PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) serves as an effective storage buffer for many SUPT16H antibodies .

Sample preparation should include complete denaturation with reducing agents and appropriate heat treatment to expose epitopes. For cell and tissue lysates, inclusion of phosphatase and deacetylase inhibitors may be critical, particularly when studying post-translational modifications like the acetylation at lysine 674 (K674) of the middle domain, which involves TIP60 histone acetyltransferase . When analyzing SUPT16H in complex with other proteins, gentler lysis conditions may be necessary to preserve protein-protein interactions. Positive controls should include known SUPT16H-expressing tissues such as mouse brain tissue, which has been validated for Western blot applications .

What experimental approaches can effectively investigate SUPT16H acetylation and its functional implications?

Investigating SUPT16H acetylation requires specialized methodology targeting post-translational modifications. The primary approach involves immunoprecipitation using anti-acetyl lysine antibodies followed by SUPT16H immunoblotting. This technique has successfully detected SUPT16H acetylation across multiple cell lines including HEK293T, HeLa, Jurkat, and NK-92 . To investigate the enzymes responsible for SUPT16H acetylation, researchers can employ TIP60-specific inhibitors like MG149, which reduces SUPT16H acetylation without significant cytotoxicity .

For mapping specific acetylation sites, researchers should generate domain-specific constructs (NTD, DD, MD, CTD) with epitope tags for expression in mammalian cells. Reciprocal immunoprecipitation assays between acetylation and tag-specific antibodies can identify which domains undergo acetylation - research has identified the middle domain (MD) as the primary acetylation target . To further characterize functional implications, researchers can examine protein-protein interactions affected by acetylation status, as exemplified by the finding that TIP60 knockdown abolishes the interaction between SUPT16H MD domain and BRD4 . Site-directed mutagenesis of lysine 674 can provide direct evidence of this residue's functional significance in acetylation-dependent molecular interactions.

How can SUPT16H antibodies be employed to investigate chromatin-associated functions?

Investigating SUPT16H's chromatin-associated functions requires specialized techniques beyond standard antibody applications. Chromatin immunoprecipitation (ChIP) assays using validated SUPT16H antibodies can map genomic binding sites and reveal associations with specific gene regulatory elements. While standard ChIP protocols work for many nuclear proteins, optimization for SUPT16H might require adjusting crosslinking conditions and sonication parameters to efficiently capture transient chromatin interactions characteristic of chromatin remodeling factors.

For studying SUPT16H's role in transcriptional regulation, researchers can combine ChIP with RNA-seq or RT-qPCR to correlate SUPT16H genomic localization with transcriptional outcomes. Evidence indicates that SUPT16H influences the expression of Notch signaling components critical for hematopoietic stem cell development . To investigate functional relationships with other epigenetic regulators, co-immunoprecipitation followed by mass spectrometry can identify SUPT16H-associated proteins. This approach has revealed that SUPT16H-BRD4 complexes associate with epigenetic silencing enzymes like EZH2 and HDAC1, contributing to gene suppression mechanisms . Sequential ChIP (re-ChIP) can determine whether these factors co-occupy the same genomic regions, providing insights into chromatin-based regulatory mechanisms.

What is the role of SUPT16H in hematopoietic stem cell development and how can antibodies help investigate this process?

SUPT16H plays a crucial role in hematopoietic stem and progenitor cell (HSPC) specification and development. Research in zebrafish has demonstrated that supt16h mutants express reduced levels of Notch-signaling components, which are essential for HSPC development . This reduction occurs due to abrogated transcription, indicating SUPT16H's importance in transcriptional regulation during hematopoiesis.

To investigate this developmental process, researchers can employ SUPT16H antibodies in several strategic approaches. Immunohistochemistry using validated SUPT16H antibodies can visualize protein expression in developing hematopoietic tissues, such as human tonsillitis and spleen tissues, where positive detection has been confirmed . Western blotting can quantify SUPT16H expression levels during different developmental stages or compare expression between wild-type and mutant models. For mechanistic studies, immunoprecipitation using SUPT16H antibodies followed by mass spectrometry can identify developmental stage-specific protein interaction partners, revealing how SUPT16H-containing complexes evolve during hematopoiesis.

Additionally, researchers investigating p53-mediated regulation of HSPC specification should note that SUPT16H and p53 coordinate to activate transcription of Notch genes during this process . Chromatin immunoprecipitation using SUPT16H antibodies can map genomic binding sites near Notch-regulated genes to establish direct regulatory relationships.

How does SUPT16H contribute to interferon signaling pathways and inflammatory responses?

SUPT16H significantly influences interferon (IFN) signaling and inflammatory gene expression. Research demonstrates that knockdown or pharmacological inhibition of SUPT16H causes upregulation of several interleukin genes, including IL-4, IL-6, and IL-8, in both HEK293T and NK-92 cells . Furthermore, SUPT16H depletion enhances luciferase expression driven by interferon-stimulating responsive element (ISRE) and interferon-gamma activated site (GAS), which mediate activation of type I (IFNα/β) and type II (IFNγ) interferon responses, respectively .

To investigate SUPT16H's role in these inflammatory pathways, researchers can implement multiple antibody-based approaches. ChIP-seq using SUPT16H antibodies can identify genomic binding sites near cytokine genes and interferon-responsive elements. Co-immunoprecipitation experiments can determine whether SUPT16H interacts with key transcription factors in the interferon signaling pathway, such as STATs or IRFs. Western blotting can assess how SUPT16H levels correlate with activation of interferon signaling components following stimulation with type I or II interferons.

For advanced mechanistic studies, researchers should consider that SUPT16H-BRD4 complexes associate with epigenetic silencing enzymes like EZH2 and HDAC1 , which may contribute to the suppression of inflammatory genes under basal conditions. Pharmacological inhibition of SUPT16H using compounds like curaxin 137 (CBL0137) provides another experimental approach to examine SUPT16H's immunoregulatory functions .

What are common challenges in SUPT16H antibody specificity and how can they be addressed?

Confirming SUPT16H antibody specificity presents several challenges that require methodical validation approaches. First, researchers should confirm the expected molecular weight - while SUPT16H has a calculated molecular weight of 120 kDa, it typically appears at approximately 140 kDa on immunoblots , likely due to post-translational modifications or structural features affecting migration. Second, researchers should perform siRNA/shRNA knockdown experiments to demonstrate specificity, as has been validated in previous studies .

Cross-reactivity with similar proteins presents another potential challenge. While SUPT16H forms a heterodimer with SSRP1 in the FACT complex, antibodies should discriminate between these partners. The search results indicate that available antibodies can effectively distinguish SUPT16H from SSRP1, as demonstrated by acetylation studies where "acetylation of SUPT16H, but not SSRP1, can be readily detected in all tested cell lines" .

For tissue-specific applications, researchers should be aware that validated positive controls include mouse brain tissue (for Western blot and immunoprecipitation) and human tonsillitis and spleen tissues (for immunohistochemistry) . When using new antibodies or examining different tissue types, peptide competition assays and comparison of multiple antibodies targeting different epitopes can provide additional validation of specificity.

How can researchers resolve inconsistent results when using SUPT16H antibodies in different experimental systems?

Inconsistent results across experimental systems when using SUPT16H antibodies may stem from several factors that researchers should systematically address. First, consider protein expression levels and post-translational modifications, which vary by cell type and physiological state. SUPT16H undergoes acetylation at lysine 674 involving TIP60 histone acetyltransferase , which could affect epitope accessibility in different cellular contexts.

Second, evaluate protocol variations that impact antibody performance. For immunohistochemistry, antigen retrieval methods significantly influence results - TE buffer pH 9.0 is recommended for SUPT16H detection, with citrate buffer pH 6.0 as an alternative . For Western blotting, transfer conditions should be optimized for high-molecular-weight proteins like SUPT16H (approximately 140 kDa) .

Third, consider the specific antibody characteristics and target regions. Different antibodies target distinct SUPT16H domains - some target internal regions (like ABIN190913 targeting DLTNKEGKKPEEK) , while others target specific amino acid regions (608-715, 187-282, C-terminus) . Domain-specific antibodies may perform differently depending on protein conformation or complex formation in various experimental systems.

To resolve inconsistencies, researchers should implement a comparative approach using multiple antibodies targeting different SUPT16H epitopes and standardize protocols across systems. If discrepancies persist, orthogonal techniques like mass spectrometry can provide antibody-independent confirmation of SUPT16H presence and modifications.

How might SUPT16H antibodies contribute to understanding p53-mediated chromatin regulation?

SUPT16H antibodies offer promising tools for investigating the emerging relationship between SUPT16H and p53-mediated chromatin regulation. Research in zebrafish has revealed that p53 influences SUPT16H function in regulating Notch gene expression during hematopoietic stem cell specification . Specifically, p53 impacts the expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes . This intricate regulatory network, where "p53 and Supt16h coordinate to activate the transcription of Notch genes during HSPC specification" , presents an exciting frontier for chromatin biology research.

To explore this p53-SUPT16H axis, researchers can employ ChIP-seq using SUPT16H antibodies in both wild-type and p53-mutant or p53-depleted systems to map genome-wide changes in SUPT16H chromatin occupancy. Sequential ChIP (re-ChIP) with p53 and SUPT16H antibodies can identify genomic regions co-occupied by both factors. Co-immunoprecipitation experiments can determine whether SUPT16H and p53 physically interact and how this interaction might be regulated by cellular stress or developmental signals. Furthermore, proximity ligation assays using validated SUPT16H and p53 antibodies can visualize and quantify their interactions in situ.

These approaches could reveal how p53, a master regulator of cellular stress responses, interfaces with SUPT16H-mediated chromatin accessibility to coordinate developmental processes and maintain genomic integrity.

What potential exists for SUPT16H antibodies in cancer research and therapeutic development?

SUPT16H antibodies hold significant potential for advancing cancer research through multiple avenues. The FACT complex, comprising SUPT16H and SSRP1, plays critical roles in transcriptional regulation and chromatin dynamics, processes frequently dysregulated in cancer. SUPT16H's interactions with epigenetic silencing enzymes EZH2 and HDAC1 , both implicated in various malignancies, suggest its potential involvement in cancer-related epigenetic reprogramming.

Research applications include using SUPT16H antibodies for immunohistochemical analysis of tumor specimens to correlate expression levels with clinical outcomes, disease progression, or treatment response. Comparative studies across cancer types could reveal tissue-specific dysregulation patterns. Furthermore, investigating SUPT16H's acetylation status in cancer cells may provide insights into altered chromatin regulation mechanisms, as TIP60 (which acetylates SUPT16H at K674) has tumor suppressor functions in multiple cancers.

From a therapeutic perspective, SUPT16H's druggability has been demonstrated through compounds like curaxin 137 (CBL0137) , which modulates its activity. SUPT16H antibodies can serve as essential tools for validating target engagement and mechanism of action studies for such compounds. Additionally, the finding that SUPT16H modulates interferon signaling pathways suggests potential implications for cancer immunotherapy research, where markers of interferon responsiveness correlate with treatment outcomes.

How can SUPT16H antibodies facilitate investigation of chromatin dynamics in stem cell differentiation and reprogramming?

SUPT16H antibodies provide valuable tools for investigating chromatin dynamics during stem cell differentiation and reprogramming processes. The critical role of SUPT16H in hematopoietic stem cell specification through Notch signaling regulation suggests it may similarly influence other stem cell lineages and differentiation pathways. Researchers can use SUPT16H antibodies to track protein localization and chromatin association throughout differentiation trajectories.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using SUPT16H antibodies can map genome-wide binding patterns across differentiation time points, revealing dynamic regulatory sites. This approach, combined with assay for transposase-accessible chromatin sequencing (ATAC-seq), can correlate SUPT16H occupancy with changes in chromatin accessibility during cellular state transitions. Additionally, CUT&RUN or CUT&Tag techniques using SUPT16H antibodies offer higher resolution and lower background alternatives to traditional ChIP for mapping chromatin interactions.

To investigate SUPT16H's functional role in reprogramming, researchers can conduct immunofluorescence microscopy using SUPT16H antibodies to visualize its nuclear distribution patterns during induced pluripotent stem cell generation. Co-immunoprecipitation experiments can identify differentiation stage-specific SUPT16H binding partners, potentially revealing context-dependent complexes that regulate cell fate decisions. Finally, researchers should consider that while global chromatin accessibility in supt16h mutants is not substantially altered, specific increase in p53 accessibility occurs , suggesting targeted rather than global effects on chromatin structure that warrant detailed investigation.