HTATSF1 Antibody

What is HTATSF1 and why is it important in research?

HTATSF1 (HIV-1 Tat specific factor 1) functions as a cofactor for the stimulation of transcriptional elongation by HIV-1 Tat, which binds to the HIV-1 promoter through Tat-TAR interaction. It may also serve as a dual-function factor that couples transcription and splicing to facilitate their reciprocal activation . Research on HTATSF1 is critical for understanding HIV pathogenesis and potentially developing therapeutic approaches. The protein has been shown to have alternatively spliced transcript variants, suggesting complex regulatory mechanisms that warrant further investigation . Due to its role in HIV-1 transcription, HTATSF1 has become an important target for researchers studying viral-host interactions and transcriptional regulation.

What are the key specifications of commercially available HTATSF1 antibodies?

HTATSF1 antibodies are typically available as polyclonal antibodies raised in rabbits . For instance, the 20805-1-AP antibody shows reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species . The calculated molecular weight of HTATSF1 is approximately 86 kDa (755 amino acids), but the observed molecular weight in experimental conditions is typically 130-140 kDa, likely due to post-translational modifications . These antibodies are generally available in liquid form, stored in PBS with sodium azide and glycerol, and should be maintained at -20°C for optimal stability . Most commercial preparations are purified using antigen affinity methods, ensuring specificity for the target protein .

Which experimental applications are supported by HTATSF1 antibodies?

HTATSF1 antibodies support multiple experimental applications, with validated protocols for:

For optimal results in immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is recommended, although citrate buffer pH 6.0 can be used as an alternative . The versatility across multiple applications makes these antibodies valuable for complementary experimental approaches to validate findings.

How should HTATSF1 antibody dilutions be optimized for Western blot applications?

For Western blot optimization with HTATSF1 antibodies, begin with a moderate dilution (1:2000) from the recommended range (1:1000-1:4000) . Prepare a dilution series (e.g., 1:1000, 1:2000, 1:4000) to determine optimal signal-to-noise ratio. Control samples should include cell lines with known HTATSF1 expression such as HeLa or HepG2 cells, which have been validated for positive detection .

When preparing samples, note that the observed molecular weight (130-140 kDa) differs significantly from the calculated weight (86 kDa) . This discrepancy could impact band identification and should be considered when interpreting results. For accurate molecular weight determination, include appropriate molecular weight markers alongside your samples.

For challenging samples or weak signals, extended exposure times may be necessary, but always compare with positive controls to differentiate between specific and non-specific signals. Methodological adjustments such as longer blocking times (1-2 hours) or overnight primary antibody incubation at 4°C may improve detection quality for low abundance targets .

What are the recommended protocols for immunoprecipitation using HTATSF1 antibodies?

For successful immunoprecipitation of HTATSF1, the following methodological approach is recommended:

• Prepare cell lysates in an appropriate lysis buffer (e.g., 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, pH 8.0)

• Use 0.5-4.0 μg of HTATSF1 antibody per 1.0-3.0 mg of total protein lysate

• Incubate lysates with antibody at 4°C for 2 hours with gentle rocking

• Add protein A Sepharose beads and incubate for an additional 0.5-1 hour

• Collect immune complexes by centrifugation and wash 3 times with lysis buffer

• Elute bound proteins using SDS-PAGE loading buffer containing a reducing agent like DTT (2.5% w/v)

HepG2 cells have been validated for positive IP detection and serve as an excellent positive control . For co-immunoprecipitation studies investigating protein-protein interactions, consider crosslinking approaches to stabilize transient complexes. When analyzing results, remember that HTATSF1 has a higher observed molecular weight (130-140 kDa) than calculated (86 kDa) , which should be considered when identifying bands.

What considerations are important for immunohistochemistry with HTATSF1 antibodies?

When performing immunohistochemistry with HTATSF1 antibodies, several methodological considerations are critical:

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary method, although citrate buffer at pH 6.0 can serve as an alternative . The antigen retrieval step is crucial due to the crosslinking effects of formalin fixation.

• Dilution optimization: Start with mid-range dilutions (approximately 1:100) from the recommended range (1:20-1:200) and optimize based on signal intensity and background levels.

• Positive control tissues: Human breast cancer tissue and human brain tissue have been validated for positive IHC detection . Include these controls to verify antibody performance.

• Incubation conditions: For weak signals, consider overnight primary antibody incubation at 4°C rather than shorter incubations at room temperature.

• Counterstaining: A light hematoxylin counterstain allows for better visualization of tissue architecture without obscuring antibody-specific signals.

• Section thickness: For optimal penetration of antibodies, tissue sections should be 4-6 μm thick.

Successful IHC staining requires careful optimization for each tissue type, and protocol modifications may be necessary for different fixation methods or tissue sources .

How can HTATSF1 antibodies be used to investigate HIV-1 transcriptional regulation?

HTATSF1 antibodies provide valuable tools for investigating HIV-1 transcriptional regulation through several advanced approaches:

• Chromatin immunoprecipitation (ChIP): Utilize HTATSF1 antibodies to immunoprecipitate chromatin fragments and identify DNA binding sites within the HIV-1 LTR region. Combine with qPCR or sequencing to quantify binding at specific regulatory elements. While not explicitly validated for ChIP in the provided data, the IP-validated antibodies can be tested for this application .

• Co-immunoprecipitation studies: Investigate interactions between HTATSF1 and other transcriptional regulators such as HIV-1 Tat, CDK9, cyclin T1, or components of the transcription/splicing machinery. This approach can reveal mechanisms of HTATSF1-mediated transcriptional elongation enhancement .

• Proximity ligation assays: Visualize and quantify protein-protein interactions between HTATSF1 and other factors in situ using antibody-based proximity detection.

• Immunofluorescence co-localization: Combine HTATSF1 antibodies with antibodies against other transcriptional regulators to assess their spatial relationship in cells, particularly under conditions of HIV-1 infection or latency reactivation .

• RNA immunoprecipitation: Investigate the RNA-binding properties of HTATSF1, particularly its association with HIV-1 transcripts, to understand its role in coordinating transcription and splicing .

For all these applications, appropriate controls, including HTATSF1 knockdown or knockout samples, should be incorporated to validate specificity .

What approaches can be used to study HTATSF1 in virus-host interactions beyond HIV-1?

While HTATSF1 was initially characterized in the context of HIV-1 transcription, emerging research suggests broader roles in virus-host interactions that can be investigated using HTATSF1 antibodies:

• Comparative studies across viral systems: Apply immunoprecipitation and Western blot analysis to examine HTATSF1 recruitment during infection with different viruses, such as henipaviruses, which have been shown to interact with host transcription factors . Protocol optimization may be necessary for different viral systems.

• Protein complex analysis: Use co-immunoprecipitation followed by mass spectrometry to identify novel viral and cellular interaction partners of HTATSF1 under different infection conditions . This approach can reveal unexpected functions beyond the established role in HIV-1 transcription.

• Subcellular localization analysis: Track changes in HTATSF1 localization during different stages of viral infection using immunofluorescence microscopy with validated antibodies for IF/ICC applications .

• Functional studies with viral protein expression: Transfect cells with expression vectors for individual viral proteins and assess their impact on HTATSF1 expression, modification, or localization using Western blot and immunofluorescence approaches .

• HTATSF1 manipulation in viral replication assays: Combine antibody-based detection methods with HTATSF1 knockdown/knockout systems to assess the impact on replication of different viruses, using the antibodies to confirm successful target depletion .

These approaches require careful experimental design and appropriate controls, but can reveal novel insights into the broader roles of HTATSF1 in viral pathogenesis beyond HIV-1.

How can HTATSF1 antibodies be utilized in studies of transcription-splicing coupling?

HTATSF1 is proposed to function as a dual-function factor that couples transcription and splicing . To investigate this complex interplay, researchers can implement several sophisticated approaches using HTATSF1 antibodies:

• Sequential chromatin immunoprecipitation (ChIP-reChIP): First immunoprecipitate with antibodies against RNA polymerase II, then perform a second immunoprecipitation with HTATSF1 antibodies to identify genomic regions where both factors co-localize, indicating sites of active transcription-splicing coupling.

• RNA-protein immunoprecipitation (RIP): Use HTATSF1 antibodies to immunoprecipitate protein-RNA complexes, followed by RNA sequencing to identify transcripts associated with HTATSF1, providing insights into its RNA-binding properties and potential splicing targets .

• Immunoprecipitation-mass spectrometry: Identify proteins that co-immunoprecipitate with HTATSF1 under different cellular conditions (e.g., stress, differentiation) to map dynamic interaction networks involved in transcription-splicing coordination .

• Alternative splicing analysis: Combine HTATSF1 knockdown/overexpression with RNA-seq and validate protein-level changes using Western blot with HTATSF1 antibodies to assess the impact on alternative splicing patterns .

• Super-resolution microscopy: Utilize immunofluorescence with HTATSF1 antibodies in combination with markers of transcription and splicing machineries to visualize nuclear co-localization at sub-diffraction resolution .

These approaches require careful optimization but can provide mechanistic insights into how HTATSF1 coordinates transcription and splicing processes in both physiological and pathological contexts.

How should researchers address discrepancies between calculated and observed molecular weights of HTATSF1?

The significant difference between the calculated molecular weight of HTATSF1 (86 kDa) and its observed molecular weight (130-140 kDa) in experimental conditions presents an important consideration for data interpretation. To address this discrepancy, researchers should:

• Verify antibody specificity: Confirm that the observed band represents HTATSF1 through additional validation methods such as:

  • HTATSF1 knockdown/knockout experiments to demonstrate band disappearance

  • Protein overexpression to demonstrate band enhancement

  • Use of multiple antibodies targeting different epitopes of HTATSF1

• Investigate post-translational modifications: The higher molecular weight may result from modifications such as:

  • Phosphorylation (multiple sites could significantly increase apparent weight)

  • SUMOylation or ubiquitination

  • Glycosylation

  Treat samples with appropriate enzymes (phosphatases, deglycosylation enzymes) and observe if this reduces the molecular weight to the calculated value.

• Assess protein-protein interactions: Strong interactions resistant to SDS denaturation might contribute to altered migration. Try more stringent sample preparation conditions or crosslinking studies to investigate this possibility.

• Compare across different cell types: The 130-140 kDa band has been observed in multiple cell lines and tissues (HeLa, HepG2, Jurkat, brain tissue) , suggesting this is the consistent form of the protein rather than a cell-specific artifact.

This molecular weight discrepancy appears to be consistent across experimental systems and likely represents biological reality rather than technical artifact .

What controls should be included when working with HTATSF1 antibodies?

Rigorous experimental design with appropriate controls is essential for reliable results when working with HTATSF1 antibodies:

• Positive controls:

  • For Western blot: Include lysates from cells with known HTATSF1 expression (HeLa, HepG2, Jurkat)

  • For IHC: Include human breast cancer or brain tissue sections known to express HTATSF1

  • For IF: Include HeLa cells which have been validated for positive detection

• Negative controls:

  • Primary antibody omission control to assess secondary antibody specificity

  • Isotype control (rabbit IgG) at equivalent concentration to assess non-specific binding

  • HTATSF1 knockdown or knockout samples, if available, to confirm antibody specificity

• Peptide competition/blocking controls: Pre-incubate the antibody with the immunogen peptide to demonstrate signal specificity. The immunogen sequence information provided (1-350 aa encoded by BC009896) can be used to design appropriate blocking peptides .

• Cross-reactivity assessment: When working across species, validate antibody performance in each target species (human, mouse, rat) as predicted reactivity may differ from actual performance .

• Loading controls: For Western blots, include appropriate loading controls (β-actin, GAPDH) to normalize protein levels and facilitate accurate quantification.

Including these controls in experimental design will enhance data reliability and facilitate accurate interpretation of results when working with HTATSF1 antibodies.

How can researchers optimize signal detection for low-abundance HTATSF1 expression?

When investigating tissues or cell lines with low HTATSF1 expression levels, several methodological optimizations can enhance detection sensitivity:

• Western blot optimization:

  • Increase protein loading (up to 50-100 μg total protein per lane)

  • Use higher antibody concentration (1:1000 rather than 1:4000)

  • Employ enhanced chemiluminescence (ECL) substrates designed for high sensitivity

  • Consider using PVDF membranes rather than nitrocellulose for better protein retention

  • Extend exposure times, using incremental exposures to find optimal signal-to-noise ratio

• Immunoprecipitation enrichment:

  • Use IP to concentrate HTATSF1 from larger sample volumes before Western blot analysis

  • Scale up antibody amount to 4.0 μg per sample to maximize target capture

• Immunohistochemistry enhancement:

  • Use higher antibody concentration (1:20 rather than 1:200)

  • Employ signal amplification systems (e.g., tyramide signal amplification)

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize antigen retrieval conditions, comparing TE buffer pH 9.0 versus citrate buffer pH 6.0

• Immunofluorescence improvements:

  • Use higher antibody concentration (1:10)

  • Employ confocal microscopy with z-stacking to collect maximum signal

  • Consider signal amplification through secondary antibody layering or fluorophore-conjugated tyramide

• Sample preparation considerations:

  • Minimize time between sample collection and fixation/lysis

  • Include protease and phosphatase inhibitors in all buffers

  • Avoid repeated freeze-thaw cycles of samples and antibody

These optimizations should be implemented systematically, changing one variable at a time while maintaining appropriate controls to ensure that enhanced signals remain specific to the target protein.

How is HTATSF1 being studied in the context of viral particle assembly?

Recent research has begun exploring HTATSF1's potential involvement in viral particle assembly beyond its established role in transcriptional regulation. Studies involving henipavirus matrix proteins have identified host factors that interact with viral M proteins and contribute to viral particle assembly . While HTATSF1 is primarily known for its role in HIV-1 transcription, similar methodological approaches could be applied to investigate its potential role in viral assembly:

• Viral-like particle (VLP) production assays: Researchers can manipulate HTATSF1 expression (through knockdown or overexpression) and measure the effects on VLP production using techniques similar to those described in the literature . HTATSF1 antibodies can be used to confirm knockdown efficiency or overexpression levels through Western blot analysis.

• Co-localization studies with viral structural proteins: Using immunofluorescence with HTATSF1 antibodies (validated for IF/ICC at 1:10-1:100 dilutions) in combination with antibodies against viral structural proteins to assess potential recruitment to assembly sites.

• Protein-protein interaction studies: Applying co-immunoprecipitation approaches using HTATSF1 antibodies (0.5-4.0 μg for IP) to identify interactions with viral structural proteins, particularly during late stages of viral replication.

• Subcellular fractionation: Combining this technique with Western blot analysis using HTATSF1 antibodies to track potential redistribution of HTATSF1 during viral assembly.

These methodological approaches could reveal novel functions of HTATSF1 in the viral life cycle beyond transcriptional regulation, potentially identifying new therapeutic targets for antiviral development.

What emerging techniques can enhance HTATSF1 protein interaction studies?

Emerging technologies are expanding the toolkit for studying HTATSF1 interactions with other cellular and viral proteins:

• Proximity-dependent biotin identification (BioID): Fusing a biotin ligase to HTATSF1 allows biotinylation of proximal proteins, which can then be purified and identified by mass spectrometry. Validate findings using co-immunoprecipitation with HTATSF1 antibodies .

• CRISPR/Cas9 genome editing for endogenous tagging: Creating knock-in cell lines with endogenous HTATSF1 tagged with small epitopes or fluorescent proteins. HTATSF1 antibodies can validate these models by comparing expression patterns and molecular weights with unmodified cells .

• Single-molecule imaging techniques: Using fluorescently labeled HTATSF1 antibodies for super-resolution microscopy or single-particle tracking to study dynamic interactions and localizations of HTATSF1 at the nanoscale level .

• Protein complementation assays: Split reporter systems (such as NanoBiT or split GFP) can be combined with immunofluorescence using HTATSF1 antibodies to correlate protein complementation signals with endogenous HTATSF1 localization .

• Quantitative interactome analysis: Using HTATSF1 antibodies for immunoprecipitation combined with SILAC or TMT labeling and mass spectrometry to quantify changes in the HTATSF1 interactome under different experimental conditions .

These advanced methodologies, when combined with validated HTATSF1 antibodies, can provide unprecedented insights into the dynamic interactions and functions of HTATSF1 in both normal cellular processes and pathological conditions such as viral infections.