TAF15 Antibody, FITC conjugated

What is TAF15 and why is it important in cellular research?

TAF15 (TATA-box binding protein associated factor 15) is a multifunctional nuclear protein with a canonical length of 592 amino acid residues and a mass of 61.8 kDa in humans. It localizes in both the nucleus and cytoplasm, with up to two different isoforms reported. As a member of the RRM TET protein family, TAF15 plays crucial roles in transcription regulation and RNA splicing mechanisms . TAF15's importance extends to cancer research, where a tumor-specific variant has been identified on the plasma membrane of stomach cancer and melanoma cells, suggesting its involvement in malignant processes . Furthermore, TAF15 has been identified as radiation-inducible in non-small cell lung cancer (NSCLC), with its overexpression correlating with worsened patient survival .

What are the typical applications for FITC-conjugated TAF15 antibodies?

FITC-conjugated TAF15 antibodies are particularly valuable for fluorescence-based detection methods. The primary applications include:

These applications leverage the fluorescent properties of FITC conjugation to directly visualize TAF15 expression and localization without the need for secondary antibody incubation steps .

How does FITC conjugation affect antibody performance compared to unconjugated antibodies?

FITC conjugation provides direct fluorescent detection capability but may influence certain performance characteristics compared to unconjugated antibodies:

What are the optimal fixation and permeabilization protocols for TAF15 detection with FITC-conjugated antibodies?

The effectiveness of TAF15 detection using FITC-conjugated antibodies is highly dependent on proper fixation and permeabilization protocols:

For immunohistochemistry applications, antigen retrieval methods significantly affect TAF15 detection. Data indicates that TE buffer at pH 9.0 provides optimal results, although citrate buffer at pH 6.0 can serve as an effective alternative . For subcellular localization studies, it's important to note that TAF15 has both nuclear and cytoplasmic distribution, requiring permeabilization protocols that maintain nuclear membrane integrity while allowing antibody access.

How can researchers optimize signal-to-noise ratio when using FITC-conjugated TAF15 antibodies?

Achieving optimal signal-to-noise ratios with FITC-conjugated TAF15 antibodies requires attention to several methodological factors:

• Titration optimization: Conduct antibody dilution series (typically starting from 1:200 to 1:800) to determine the optimal concentration that maximizes specific signal while minimizing background fluorescence .

• Blocking protocol refinement: Implement a robust blocking step using 5-10% normal serum from the same species as the secondary antibody (if used in a detection system) or BSA to minimize non-specific binding.

• Autofluorescence countermeasures: For tissues with high autofluorescence (particularly formalin-fixed specimens), consider:

  • Pre-treatment with 0.1% Sudan Black B in 70% ethanol

  • Short incubation in 0.1% sodium borohydride solution

  • Using appropriate filter sets to distinguish FITC signal from autofluorescence

• Photobleaching prevention: Incorporate anti-fade mounting media containing DAPI for nuclear counterstaining while preserving FITC fluorescence during imaging and storage.

These methodological refinements can significantly enhance the detection of both standard nuclear/cytoplasmic TAF15 and the tumor-specific membrane variants identified in certain cancer types .

How can TAF15 antibodies be applied to investigate its role in cancer progression?

Research indicates that TAF15 plays significant roles in cancer biology, offering several strategic applications for antibody-based investigations:

• Surface expression analysis: Flow cytometry and immunofluorescence microscopy with FITC-conjugated TAF15 antibodies can identify the tumor-specific 78-kDa TAF15 variant expressed on the plasma membrane of stomach cancer and melanoma cells, which is absent in healthy tissues .

• Prognostic biomarker development: Immunohistochemical studies have shown that TAF15 overexpression correlates with worsened survival in NSCLC patients. Standardized protocols using calibrated antibody dilutions (1:250-1:1000) can quantify expression levels for correlation with clinical outcomes .

• Radiation response mechanisms: TAF15 has been identified as radiation-inducible in cancer, with surface expression increasing following radiotherapy. Dual-staining approaches combining FITC-conjugated TAF15 antibodies with markers of radiation damage can elucidate the temporal relationship between radiation exposure and TAF15 expression dynamics .

• Functional interference studies: Based on findings that the PAT-BA4 antibody against TAF15 inhibits tumor cell motility and adhesion, researchers can employ antibody-based interventions to probe TAF15's mechanistic contributions to metastatic potential .

What are the considerations for using TAF15 antibodies in RNA immunoprecipitation (RIP) studies?

TAF15's role in RNA processing makes RNA immunoprecipitation (RIP) a particularly valuable application for TAF15 antibodies. Key methodological considerations include:

• Crosslinking optimization: Due to TAF15's roles in both transcription and RNA splicing, researchers should consider:

  • Formaldehyde crosslinking (1% for 10 minutes) for protein-DNA interactions

  • UV crosslinking (254nm, 400 mJ/cm²) for direct RNA-protein interactions

• Antibody selection criteria: For RIP applications, prioritize antibodies validated specifically for immunoprecipitation efficiency. Published research has successfully employed TAF15 antibodies for RIP applications, with particular success reported for rabbit polyclonal antibodies targeting TAF15 .

• Control implementation: Include:

  • Input controls (pre-immunoprecipitation sample)

  • IgG isotype controls to establish background binding

  • Known TAF15-associated transcripts as positive controls

• RNA integrity preservation: Incorporate RNase inhibitors throughout the protocol and optimize lysis conditions to maintain RNA integrity while still releasing nuclear TAF15-RNA complexes.

RIP studies with TAF15 antibodies have contributed to understanding its role in post-transcriptional regulation mechanisms that may influence cancer progression and radiation response .

How can researchers address TAF15 isoform specificity in their experimental design?

TAF15 exists in multiple isoforms, with up to two different variants reported in humans. Researchers seeking isoform-specific information should consider:

• Epitope mapping analysis: Review the immunogen information for the antibody to determine whether it targets regions common to all isoforms or isoform-specific epitopes. The TAF15 fusion protein Ag20828 serves as the immunogen for several commercially available antibodies .

• Western blot validation: Perform western blot analysis with positive controls of known isoform expression (HEK-293 cells, HeLa cells) to verify the antibody's detection pattern. The canonical form appears at approximately 62 kDa, while the observed molecular weight is typically 68 kDa. The tumor-specific variant has been reported at 78 kDa .

• Knockout/knockdown controls: Include TAF15 knockout or knockdown samples to confirm antibody specificity, particularly in studies examining the tumor-specific variant. Several published studies have successfully employed TAF15 KD/KO approaches for antibody validation .

• Mass spectrometry correlation: For definitive isoform identification, complement antibody-based studies with mass spectrometry analysis of immunoprecipitated material to precisely identify the detected isoforms.

What are the common challenges in detecting TAF15 using fluorescently-labeled antibodies?

Researchers frequently encounter several technical challenges when working with FITC-conjugated TAF15 antibodies:

• Nuclear penetration limitations: TAF15's predominant nuclear localization can present accessibility challenges. To overcome this:

  • Extend permeabilization times (15-20 minutes with 0.2% Triton X-100)

  • Consider heat-mediated antigen retrieval even for cell preparations

  • Optimize fixation to prevent overfixation that might mask nuclear antigens

• Distinguishing specific patterns from artifacts: TAF15 exhibits both diffuse and punctate nuclear staining alongside cytoplasmic localization. Validation approaches should include:

  • Comparison with unconjugated primary antibody and labeled secondary detection systems

  • Co-staining with other nuclear markers to confirm nuclear compartment identification

  • Z-stack imaging to distinguish true signal from optical artifacts

• Tumor-specific variant detection: The reported 78-kDa tumor-specific membrane variant requires specialized detection approaches:

  • Membrane-specific staining protocols without permeabilization

  • Careful titration to detect potentially lower-abundance surface expression

  • Comparison with normal tissue controls to confirm specificity

How can researchers validate the specificity of TAF15 antibody signals in multi-color fluorescence applications?

Multi-color fluorescence applications require rigorous validation to ensure signal specificity:

• Spectral overlap controls: When combining FITC-conjugated TAF15 antibodies with other fluorophores:

  • Run single-color controls to establish compensation settings

  • Consider fluorophore combinations that minimize spectral overlap with FITC

  • Implement sequential scanning for confocal microscopy applications

• Co-localization validation strategies:

  • Perform co-staining with antibodies against known TAF15 interaction partners (components of TFIID complex)

  • Use subcellular markers to confirm expected localization patterns (nuclear, cytoplasmic, or membrane in tumor cells)

  • Quantify co-localization using appropriate statistical measures (Pearson's correlation coefficient)

• Technical validation controls:

  • Include absorption controls omitting primary antibody

  • Implement blocking peptide competition assays to confirm epitope specificity

  • Use multiple antibody clones targeting different epitopes to confirm staining patterns

These validation approaches are particularly critical when examining TAF15's differential localization in normal versus cancer cells .

What methodologies can be employed to study TAF15's role in the radiation-inducible stress response?

TAF15's involvement in radiation response pathways offers several strategic approaches for investigation:

• Temporal expression analysis: Using FITC-conjugated TAF15 antibodies, researchers can establish time-course studies to track:

  • Changes in expression levels post-radiation (typical timepoints: 1h, 6h, 24h, 48h)

  • Shifts in subcellular localization following radiation exposure

  • Surface induction in cancer cell lines, particularly NSCLC models

• Co-staining with DNA damage markers: Combine TAF15 detection with markers of radiation-induced damage:

  • γH2AX for double-strand break identification

  • 53BP1 for DNA damage response pathway activation

  • Markers of cell cycle arrest (p21, cyclins) to correlate with TAF15 expression changes

• Functional inhibition studies: Employ approaches to modulate TAF15 activity:

  • siRNA knockdown before radiation exposure

  • Antibody-mediated blocking of surface TAF15 in tumor models

  • CRISPR-Cas9 engineered TAF15 mutants to identify radiation-response domains

• Downstream pathway analysis: Investigate TAF15's role in radiation response by examining:

  • Changes in RNA splicing patterns following radiation (RNA-seq)

  • Altered transcriptional profiles in TAF15-depleted cells post-radiation

  • Protein interaction networks specific to irradiated conditions (IP-MS)

How can TAF15 antibodies contribute to understanding the relationship between TAF15 and other TET family members in pathological conditions?

TAF15 belongs to the TET family of RNA-binding proteins that includes FUS and EWS, which have been implicated in various pathological conditions. Research strategies to investigate their interrelationships include:

• Co-immunoprecipitation studies: Using TAF15 antibodies validated for IP applications to:

  • Identify protein complexes containing multiple TET family members

  • Determine how these interactions change in disease states

  • Map interaction domains through deletion mutant analysis

• Comparative localization studies: Employing multi-color fluorescence with FITC-conjugated TAF15 antibodies and differently-labeled antibodies against FUS and EWS to:

  • Map subcellular distribution patterns in normal versus pathological tissues

  • Identify cell types with unique TET family expression profiles

  • Track relocalization events under stress conditions

• Functional redundancy analysis: Using antibody-based approaches to:

  • Block specific TET family members individually and in combination

  • Correlate expression patterns across family members in patient samples

  • Develop multi-target therapeutic strategies based on expression profiles

These approaches can help elucidate whether the tumor-specific TAF15 variant represents a unique pathological mechanism or if similar variants exist across the TET family in cancer contexts .

What experimental designs are recommended for investigating contradictory findings regarding TAF15 expression in different cancer types?

Research has revealed seemingly contradictory roles for TAF15 across different cancer types, requiring carefully designed studies to resolve these discrepancies:

• Standardized expression analysis protocol:

  • Implement consistent antibody dilutions (1:250-1:1000 for IHC)

  • Use automated image analysis to quantify expression levels

  • Include tissue microarrays spanning multiple cancer types for direct comparison

• Isoform-specific profiling:

  • Employ antibodies targeting common versus variable regions

  • Combine with RT-PCR to identify isoform-specific expression patterns

  • Correlate protein detection with transcript analysis across cancer types

• Functional context determination:

  • Investigate TAF15 in context of tumor microenvironment factors

  • Examine radiation response across cancer types with variable TAF15 expression

  • Correlate expression with specific oncogenic driver mutations

• Clinical correlation studies:

  • Design multi-cancer tissue studies with consistent staining protocols

  • Correlate TAF15 expression patterns with patient outcomes

  • Stratify results by cancer type, stage, and treatment history

These methodological approaches can help reconcile apparently contradictory findings related to TAF15's role in stomach cancer, melanoma, and NSCLC, where different expression patterns and functional impacts have been reported .