SUPT16H Antibody, Biotin conjugated

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

SUPT16H (also known as FACT140, FACTP140, or hSPT16) functions as the 140 kDa subunit of the FACT complex, which plays a critical role in chromatin remodeling during transcription, DNA replication, and repair. The protein contains four distinct domains: N-terminal domain (NTD), dimerization domain (DD), middle domain (MD), and C-terminal domain (CTD), each with specific functions in chromatin interaction. Recent research has demonstrated that SUPT16H is acetylated at lysine 674 (K674) in the middle domain through the action of TIP60 histone acetyltransferase . This post-translational modification mediates SUPT16H's interaction with BRD4, a key acetylation reader containing bromodomains . Together, these proteins contribute to both gene activation and, interestingly, gene suppression pathways including silencing of integrated HIV-1 proviruses .

What are the molecular characteristics of the SUPT16H protein?

SUPT16H is a 119 kDa protein (calculated molecular weight) that functions as the larger subunit of the FACT heterodimer complex . The protein is encoded by the SUPT16H gene (Gene ID: 11198) and has the UniProt accession number Q9Y5B9 . SUPT16H contains multiple functional domains that interact with chromatin components, with the middle domain (MD) being particularly important as it undergoes acetylation by TIP60 . This acetylation serves as a recognition site for BRD4 binding, highlighting the importance of post-translational modifications in regulating SUPT16H function . The protein is highly conserved across species, with antibodies showing cross-reactivity between human, mouse, and rat SUPT16H proteins .

What advantages does Biotin conjugation provide for SUPT16H antibody applications?

Biotin conjugation of SUPT16H antibody provides several methodological advantages for research applications. The strong affinity interaction between biotin and streptavidin (Kd ≈ 10^-15 M) enables signal amplification when used with enzyme-conjugated streptavidin detection systems . This amplification significantly improves sensitivity for detecting low-abundance SUPT16H protein in complex biological samples. Additionally, the biotin-streptavidin system offers flexible detection options, as researchers can select from various enzyme-conjugated streptavidin reagents (HRP, AP, fluorophores) depending on their specific experimental requirements. The recommended dilution range for Biotin-conjugated SUPT16H antibody is 1:50 to 1:1,000 when working with enzyme-conjugated streptavidin detection systems .

What are the optimal sample preparation methods for detecting SUPT16H in different cellular contexts?

For effective detection of SUPT16H in various cellular contexts, sample preparation procedures must preserve both protein integrity and post-translational modifications. When preparing nuclear extracts (where SUPT16H predominantly localizes), use gentle lysis buffers containing phosphatase inhibitors (NaF, Na3VO4) and deacetylase inhibitors (TSA, nicotinamide) to preserve acetylation at K674, which is critical for BRD4 interactions . For immunoprecipitation studies, crosslinking with 1% formaldehyde for 10 minutes at room temperature has been demonstrated to effectively capture SUPT16H-chromatin interactions. When performing protein extraction for Western blotting, include 0.1% SDS in RIPA buffer to ensure complete solubilization of chromatin-bound SUPT16H. Cell fractionation protocols should include DNase I treatment (10-50 μg/ml, 30 minutes at room temperature) to release chromatin-bound SUPT16H for complete recovery and analysis.

How should researchers design experimental controls when using SUPT16H Biotin conjugated antibody?

Implementing proper experimental controls is essential for ensuring reliable results with SUPT16H Biotin conjugated antibody. Positive controls should include cell types known to express high levels of SUPT16H, such as HEK293T, HeLa, Jurkat, or NK-92 cells, all of which have demonstrated detectable SUPT16H acetylation . For negative controls, researchers should use one of the following approaches: (1) siRNA knockdown of SUPT16H, which has been shown to increase HIV-1 LTR promoter-driven gene expression ; (2) blocking peptide experiments using the immunogen peptide from the C-terminal region of human SUPT16H ; or (3) isotype-matched control IgG from the same host species (rabbit) used at the same concentration . For biotin-specific controls, include a parallel sample treated with an irrelevant biotin-conjugated antibody of the same isotype to control for non-specific streptavidin binding.

What dilution ranges and incubation conditions are optimal for different applications?

Optimizing antibody dilutions and incubation conditions is critical for generating reproducible results across different applications. The table below summarizes recommended parameters based on compiled experimental data:

For Biotin-conjugated antibody specifically, dilutions of 1:50 to 1:1,000 are recommended when working with enzyme-conjugated streptavidin detection systems .

How can SUPT16H Biotin conjugated antibody be utilized in chromatin immunoprecipitation (ChIP) experiments?

SUPT16H Biotin conjugated antibody offers specific advantages for chromatin immunoprecipitation studies investigating genomic binding sites of the FACT complex. For optimal ChIP protocols, crosslink cells with 1% formaldehyde for 10 minutes at room temperature, followed by quenching with 125 mM glycine. After cell lysis and sonication to generate 200-500 bp DNA fragments, immunoprecipitate chromatin using the Biotin-conjugated SUPT16H antibody (1:50 dilution) bound to streptavidin magnetic beads. This approach eliminates the need for secondary antibody and protein A/G beads, reducing background and increasing specificity. Critically, include deacetylase inhibitors (5 mM sodium butyrate, 1 μM TSA) in all buffers to preserve SUPT16H acetylation, which influences its genomic distribution . When analyzing ChIP-seq data, co-mapping with BRD4 binding sites is recommended based on the demonstrated interaction between SUPT16H and BRD4 , particularly at gene regulatory regions where the SUPT16H-BRD4 complex has been shown to contribute to gene silencing.

What methodological approaches can resolve contradictory findings in SUPT16H functional studies?

Resolving contradictory findings regarding SUPT16H function requires systematic methodological approaches addressing potential confounding factors. First, implement domain-specific analysis by using antibodies targeting different SUPT16H domains (NTD, DD, MD, CTD) to distinguish domain-specific functions, as research has shown that only the MD domain is directly acetylated while the DD domain binds to other acetylated proteins . Second, evaluate post-translational modifications by comparing results obtained before and after treatment with TIP60 inhibitor MG149 or TIP60 knockdown, which significantly reduces SUPT16H acetylation and BRD4 interaction . Third, employ genetic rescue experiments using wild-type SUPT16H and the K674R acetylation-deficient mutant to definitively determine acetylation-dependent functions. Fourth, consider cell type-specific effects by comparing results across different cell lines known to express SUPT16H (HEK293T, HeLa, Jurkat, NK-92) . Finally, use complementary inhibitors including both the SUPT16H inhibitor curaxin 137 (CBL0137) and the BRD4 inhibitor JQ1, which drastically reduces SUPT16H-BRD4 interaction .

How can researchers distinguish between direct and indirect effects of SUPT16H in gene regulation experiments?

Distinguishing between direct and indirect regulatory effects of SUPT16H requires integrative experimental approaches. Implement rapid induction systems using an auxin-inducible degron (AID) tag on endogenous SUPT16H, allowing protein depletion within 30-60 minutes and enabling observation of immediate transcriptional effects before secondary responses occur. Combine this with nascent RNA sequencing (NET-seq or TT-seq) to capture ongoing transcription rather than steady-state RNA levels, providing a more direct readout of SUPT16H's transcriptional impact. Perform sequential ChIP (re-ChIP) experiments using SUPT16H Biotin conjugated antibody followed by BRD4 antibody to identify genomic loci where both factors co-occupy, as their interaction has been demonstrated to be functionally significant . Conduct in vitro transcription assays with reconstituted chromatin templates, purified FACT complex, and specific transcription factors to biochemically dissect SUPT16H's direct contribution to transcriptional processes. Implement CRISPR-based genome editing to mutate the SUPT16H K674 acetylation site to determine the specific contribution of this post-translational modification to gene regulation.

How can researchers address weak or inconsistent signals when using SUPT16H Biotin conjugated antibody?

When confronting weak or inconsistent signals with SUPT16H Biotin conjugated antibody, a systematic troubleshooting approach should be implemented. First, optimize protein extraction by using nuclear fractionation protocols with stringent conditions (0.3-0.4M NaCl buffer) to efficiently extract chromatin-bound SUPT16H, as it predominantly associates with chromatin . Second, preserve post-translational modifications by incorporating deacetylase inhibitors (1μM TSA, 5mM sodium butyrate) in all buffers since SUPT16H acetylation at K674 is critical for its detection and function . Third, enhance signal amplification by using tyramide signal amplification (TSA) systems compatible with biotin-streptavidin detection, which can increase sensitivity up to 100-fold compared to conventional detection methods. Fourth, optimize antigen retrieval for fixed samples using citrate buffer (pH 6.0) with heat treatment (95°C for 20 minutes) to expose the C-terminal epitope that the antibody recognizes . Finally, reduce background by including additional blocking steps with avidin/biotin blocking kits before antibody incubation to minimize endogenous biotin interference.

What factors affect SUPT16H detection and epitope accessibility in different experimental contexts?

Several critical factors influence SUPT16H detection and epitope accessibility across experimental platforms. Post-translational modifications significantly impact antibody recognition, particularly because acetylation of SUPT16H at lysine 674 alters its conformation and interactions . Treatment with TIP60 inhibitor MG149 or TIP60 knockdown significantly reduces SUPT16H acetylation , potentially affecting epitope accessibility. Protein-protein interactions present another challenge, as SUPT16H forms complexes with multiple partners including SSRP1 (forming the FACT complex) and BRD4 , which may mask antibody binding sites. The cellular fixation method critically affects epitope preservation, with methanol/acetone fixation (1:1) generally preserving the C-terminal epitope better than paraformaldehyde for immunocytochemistry applications. Finally, the chromatin state influences accessibility, as SUPT16H tightly associates with chromatin structures that may need to be disrupted with nucleases (25-50 U/ml DNase I, 30 minutes at 37°C) for complete extraction and detection.

How should researchers interpret cross-reactivity data for SUPT16H antibodies across species?

Interpreting cross-reactivity data for SUPT16H antibodies requires careful evaluation of sequence conservation and experimental validation. The SUPT16H Biotin conjugated antibody (based on the unconjugated version) recognizes endogenous levels of human SUPT16H protein, with predicted reactivity across multiple species including pig, bovine, horse, sheep, rabbit, and dog . This cross-reactivity is supported by the high conservation of the C-terminal region of SUPT16H across mammals. When validating cross-reactivity, researchers should implement sequence alignment analysis to determine the exact percentage of identity between human SUPT16H and the target species within the immunogen region (C-terminal domain). Western blot analysis using positive control lysates from target species is essential, with expected molecular weight verification (~119 kDa) . For ambiguous results, researchers should perform antibody pre-absorption tests with the immunogen peptide to confirm specificity. For confirmatory species validation, knockout/knockdown controls in the target species cells should be implemented to verify specificity and rule out non-specific binding.

How does SUPT16H contribute to epigenetic regulation through its interaction with BRD4?

SUPT16H plays a sophisticated role in epigenetic regulation through its newly discovered interaction with BRD4, a key acetylation reader. Research has revealed that SUPT16H is acetylated at lysine 674 (K674) of its middle domain by TIP60 histone acetyltransferase . This acetylation creates a binding site for BRD4, which contains two bromodomains capable of recognizing acetylated lysine residues . The interaction between SUPT16H and BRD4 is abolished by the bromodomain inhibitor JQ1, confirming that BRD4 recognizes SUPT16H through its acetyl lysine-binding capacity . Furthermore, knockdown of TIP60 significantly decreases SUPT16H acetylation and consequently reduces SUPT16H-BRD4 interaction . This SUPT16H-BRD4 complex contributes to gene suppression functions, including silencing of integrated HIV-1 proviruses, representing a previously uncharacterized regulatory mechanism distinct from their well-studied roles in transcriptional activation . These findings suggest a model where the acetylation-dependent SUPT16H-BRD4 interaction serves as a regulatory switch determining whether these factors function in gene activation or repression contexts.

What are the emerging applications of SUPT16H antibodies in disease-related research?

SUPT16H antibodies are increasingly being utilized to investigate disease mechanisms related to chromatin dysregulation. In cancer research, SUPT16H's role in maintaining chromatin structure during rapid cell division makes it a potential biomarker for aggressive tumors. The inhibition of SUPT16H using curaxin 137 (CBL0137) enhances HIV-1 LTR promoter activity, suggesting therapeutic applications in HIV latency reversal strategies . In neurodegenerative disease research, SUPT16H antibodies are being used to study chromatin alterations associated with aging and neuronal dysfunction. The SUPT16H-BRD4 interaction represents a potential intervention point, as BRD4 inhibitors like JQ1 disrupt this complex and are already in clinical trials for various diseases. Importantly, the ability to detect acetylated SUPT16H using specific antibodies may provide insights into TIP60 acetyltransferase activity, which is dysregulated in multiple pathological conditions. These emerging applications highlight the growing importance of SUPT16H antibodies as tools for understanding disease mechanisms and identifying potential therapeutic targets.