TAF5L Antibody

What is TAF5L and why is it important in research?

TAF5L (TBP-Associated Factors 5-Like) is a 64-66 kDa nuclear protein belonging to the TAF5 family. It functions as a component of several histone acetylase complexes including PCAF, STAGA, and TFTC. These complexes acetylate histone H3 within nucleosomes, facilitating the recognition and binding of transcriptional activators . Human TAF5L contains 589 amino acids with an N-terminal homodimerization interface (amino acids 90-198) and six consecutive WD repeats (amino acids 266-547) .

Its importance stems from its role as an epigenetic regulator essential for somatic reprogramming. Working with TAF6L, it regulates target genes through H3K9ac deposition and MYC recruitment, triggering MYC regulatory networks that orchestrate gene expression programs controlling embryonic stem cell states . Given these critical functions, TAF5L antibodies are valuable tools for investigating transcriptional regulation and epigenetic mechanisms.

What experimental applications are TAF5L antibodies suitable for?

TAF5L antibodies have been validated for multiple experimental applications:

When designing experiments, it's critical to select antibodies validated for your specific application and optimize conditions for your experimental system.

What is the cellular localization of TAF5L and how does this affect antibody selection?

TAF5L is predominantly a nuclear protein that forms part of several histone acetylase complexes . Western blot analysis with antibodies like AF6785 has demonstrated that TAF5L is primarily detected in nuclear extracts rather than cytoplasmic fractions of cell lines including RPMI 8226, 293T, and U2OS .

This nuclear localization has important implications for antibody selection and experimental design:

• For immunofluorescence studies, antibodies with proven nuclear staining patterns should be selected (such as ab172621)

• Cell fractionation protocols are recommended when performing western blot analysis to enhance detection sensitivity

• For IHC applications, antigen retrieval methods may need optimization - suggested protocols include TE buffer pH 9.0 or citrate buffer pH 6.0

• Fixation methods preserving nuclear architecture are preferable for microscopy applications

Understanding this localization pattern helps researchers design appropriate controls and interpret results accurately.

How should researchers optimize Western blot protocols for TAF5L detection?

Optimizing Western blot protocols for TAF5L detection requires attention to several key parameters:

• Sample preparation:

  • For nuclear proteins like TAF5L, proper cell fractionation is crucial

  • Load approximately 10-20 μg of nuclear extract (as demonstrated with RPMI 8226, 293T, and U2OS cell lines)

  • For whole cell lysates, successful detection has been demonstrated with HT-29, HT-1080, Jurkat, and HeLa lysates at 10 μg

• Antibody dilution optimization:

  • Start with manufacturer recommendations (e.g., 1:500-1:3000 for 19274-1-AP)

  • For AF6785, 1 μg/mL has been validated

  • For ab172621, 1:1000 dilution is recommended

• Detection conditions:

  • PVDF membranes are suitable for TAF5L detection

  • Reducing conditions are recommended (as validated with AF6785)

  • The expected band size is approximately 65-66 kDa

• Secondary antibody selection:

  • Match to the host species (e.g., HRP-conjugated Anti-Sheep IgG for AF6785, Anti-Rabbit for rabbit-derived antibodies)

This methodological approach ensures optimal detection of TAF5L while minimizing background and non-specific binding.

What are the critical considerations for immunohistochemical detection of TAF5L?

For successful immunohistochemical detection of TAF5L, researchers should consider:

• Tissue preparation and antigen retrieval:

  • Suggested antigen retrieval with TE buffer pH 9.0 is recommended

  • Alternative approach: citrate buffer pH 6.0

  • Complete antigen retrieval is critical due to TAF5L's nuclear localization and potential epitope masking

• Antibody selection and dilution:

  • For 19274-1-AP, use dilution range of 1:50-1:500

  • Titration is essential for each tissue type to optimize signal-to-noise ratio

• Validation in relevant tissues:

  • Positive IHC detection has been validated in human ovary cancer tissue

  • Include appropriate positive and negative controls

• Detection systems:

  • Choose detection systems compatible with rabbit primary antibodies (for 19274-1-AP)

  • Optimize visualization methods based on expression levels

Meticulous attention to these factors ensures specific and reproducible detection of TAF5L in tissue samples.

What cell lines are recommended as positive controls for TAF5L antibody validation?

Several cell lines have been validated as positive controls for TAF5L expression:

When validating a new TAF5L antibody or establishing detection methods in your laboratory, these cell lines serve as reliable positive controls. Jurkat cells are particularly well-validated across multiple antibodies and could be considered a primary choice for initial validation experiments .

How can researchers address inconsistent TAF5L detection in Western blot experiments?

Inconsistent TAF5L detection in Western blots may result from several factors:

• Nuclear localization issues:

  • Insufficient nuclear protein extraction may lead to weak signals

  • Solution: Implement proper cell fractionation protocols separating cytoplasmic and nuclear extracts, as demonstrated with AF6785 antibody

  • Compare loading 20 μg cytoplasmic vs. 10 μg nuclear extracts to verify localization

• Protein degradation:

  • TAF5L may be subject to proteolytic degradation

  • Solution: Use fresh samples, maintain proper cold chain, and include protease inhibitors in lysis buffers

• Antibody specificity concerns:

  • Different antibodies target distinct epitopes within TAF5L

  • Solution: AF6785 targets Met1-Tyr189 , while other antibodies target different regions; select antibodies validated for your application

• Buffer compatibility:

  • Buffer compositions affect detection sensitivity

  • Solution: For AF6785, Immunoblot Buffer Group 1 under reducing conditions has been validated

• Alternative splice variants:

  • A potential splice variant with Val substitution for aa 325-589 has been reported

  • Solution: Use antibodies targeting conserved regions if multiple isoforms are expected

Addressing these factors systematically will help resolve inconsistent detection issues.

How should researchers interpret differential TAF5L expression patterns across cellular compartments?

Interpreting differential TAF5L expression across cellular compartments requires careful analysis:

• Expected localization pattern:

  • TAF5L is predominantly nuclear, functioning in histone acetylase complexes

  • Western blot analysis typically shows stronger signals in nuclear vs. cytoplasmic fractions

• Interpreting unexpected cytoplasmic signals:

  • Could indicate:

    • Experimental artifacts (cross-reactivity)

    • Cell cycle-dependent redistribution

    • Novel biological functions

    • Protein mislocalization in disease states

• Validation approaches:

  • Confirm with multiple antibodies targeting different epitopes

  • Use immunofluorescence to visualize localization directly

  • Perform subcellular fractionation with clean markers for each compartment

  • Consider siRNA knockdown to confirm specificity

• Biological significance assessment:

  • Compare expression patterns between normal and disease states

  • Investigate correlation with functional outcomes

  • Consider context-specific regulation of TAF5L localization

This analytical framework ensures accurate interpretation of TAF5L distribution data.

How can TAF5L antibodies be utilized to investigate epigenetic regulation mechanisms?

TAF5L antibodies can be powerful tools for investigating epigenetic regulation through several sophisticated approaches:

• Chromatin Immunoprecipitation (ChIP) studies:

  • While not explicitly validated in the provided search results, TAF5L antibodies may be adaptable for ChIP

  • This would allow mapping of TAF5L genomic binding sites

  • Combined with sequencing (ChIP-seq), this approach can reveal genome-wide distribution patterns

  • Integration with H3K9ac ChIP data would provide insights into TAF5L's role in histone acetylation

• Co-immunoprecipitation for complex composition analysis:

  • TAF5L antibody 19274-1-AP has been validated for CoIP applications

  • This enables investigation of TAF5L interactions within PCAF, STAGA, and TFTC complexes

  • Can identify novel protein interactions in different cellular contexts

• Proximity ligation assays (PLA):

  • Combining TAF5L antibodies with antibodies against MYC or other transcription factors

  • This approach can visualize and quantify protein interactions in situ

  • Particularly valuable for confirming the reported role of TAF5L in MYC recruitment

• Functional studies with simultaneous detection:

  • Using TAF5L antibodies alongside markers of active transcription

  • Correlating TAF5L binding with H3K9ac deposition and transcriptional activity

  • Investigating the dynamics of these processes during cellular reprogramming

These advanced applications leverage TAF5L antibodies to gain mechanistic insights into epigenetic regulation beyond simple protein detection.

What approaches can researchers use to study TAF5L's role in somatic reprogramming?

To investigate TAF5L's role in somatic reprogramming, researchers can implement the following methodological approaches:

• Temporal expression analysis during reprogramming:

  • Using TAF5L antibodies for Western blot or immunofluorescence at different stages

  • Correlating TAF5L levels with reprogramming efficiency markers

  • Examining co-expression with TAF6L, its functional partner in reprogramming

• Knockdown/knockout with phenotypic rescue:

  • Depleting endogenous TAF5L using siRNA/CRISPR

  • Measuring effects on H3K9ac deposition using ChIP or immunofluorescence

  • Performing rescue experiments with wild-type vs. mutant TAF5L constructs

  • Using TAF5L antibodies to confirm knockdown efficiency and rescue expression levels

• Genome-wide studies integrating multiple data types:

  • ChIP-seq for TAF5L binding sites during reprogramming

  • RNA-seq to correlate binding with expression changes

  • Cut&Run or CUT&Tag as alternative approaches for mapping TAF5L genomic localization

  • Integration with H3K9ac and MYC binding data to construct regulatory networks

• Live-cell imaging with compatible antibody fragments:

  • Using fluorescently labeled Fab fragments derived from TAF5L antibodies

  • Tracking TAF5L dynamics during reprogramming process

  • Correlating with chromatin accessibility changes

• Interaction studies with pluripotency factors:

  • CoIP experiments using TAF5L antibodies to pull down complexes

  • Mass spectrometry analysis of interacting partners at different reprogramming stages

  • Investigation of OCT4, SOX2, and NANOG interactions with TAF5L

These methodologies provide complementary approaches to dissect TAF5L's mechanistic role in the complex process of somatic reprogramming.

What factors should researchers consider when selecting TAF5L antibodies for specific applications?

Selecting the optimal TAF5L antibody requires careful consideration of several factors:

For challenging applications or novel experimental systems, preliminary validation with multiple antibodies targeting different epitopes is recommended to confirm specificity and optimize performance.

How do different TAF5L antibodies compare in terms of specificity and sensitivity?

Comparing TAF5L antibodies reveals important differences in specificity and sensitivity:

• Western blot performance:

  • AF6785 (Sheep polyclonal): Demonstrates specific detection at approximately 65 kDa in nuclear extracts from RPMI 8226, 293T, and U2OS cell lines; minimal background reported

  • ab172621 (Rabbit monoclonal): Shows strong, specific bands at 66 kDa in HT-29, HT-1080, Jurkat, and HeLa lysates; monoclonal nature may provide higher specificity

  • 19274-1-AP (Rabbit polyclonal): Positively detects TAF5L in Jurkat cells with observed molecular weight of 66 kDa

• Immunohistochemistry/Immunofluorescence specificity:

  • 19274-1-AP: Validated for IHC in human ovary cancer tissue; requires optimization of antigen retrieval methods

  • ab172621: Shows clear nuclear localization in immunofluorescence studies with HeLa cells

  • DF3046: Validated for IF/ICC applications, though specific performance metrics aren't provided in the search results

• Sensitivity considerations:

  • Nuclear extraction efficiency significantly impacts sensitivity for all antibodies

  • Recommended dilution ranges vary (1:500-1:3000 for 19274-1-AP in WB; 1:1000 for ab172621)

  • Subcellular fractionation enhances detection sensitivity for nuclear TAF5L

• Cross-reactivity assessment:

  • The presence of a potential splice variant (Val substitution for aa 325-589) may affect detection patterns

  • Antibodies targeting the N-terminal region (like AF6785, Met1-Tyr189) should detect all known isoforms

This comparative analysis helps researchers select the most appropriate antibody based on their specific experimental requirements and available biological materials.