TAF4B Antibody, FITC conjugated

What is TAF4B and what are its primary functions in cellular processes?

TAF4B is a cell type-specific subunit of the general transcription factor TFIID that functions as a gene-selective coactivator in certain cells. It plays a central role in mediating promoter responses to various activators and repressors . Specifically, TAF4B:

• Acts as a transcriptional coactivator of the p65/RELA NF-kappa-B subunit

• Participates in the activation of antiapoptotic genes including TNFAIP3

• Functions in regulating folliculogenesis

• Acts as a coactivator of B-cell-specific transcription through interaction with OCBA/POU2AF1

• Plays critical roles in spermiogenesis and oogenesis

TAF4B is highly expressed in ovaries and testes and is required for mouse fertility, making it a crucial target for reproductive biology research .

What applications has the TAF4B Antibody, FITC conjugated been validated for?

According to product specifications, TAF4B FITC-conjugated antibodies have been validated for several applications:

• ELISA (recommended dilution 1:2000-1:10000)

• Immunohistochemistry (IHC) (recommended dilution 1:500-1:1000)

• Immunofluorescence (IF) (recommended dilution 1:50-1:200)

Researchers should note that optimal dilutions/concentrations should be determined by the end user based on specific experimental conditions . Validation data typically includes immunofluorescence staining of cell lines like A549 cells (at 1:166 dilution) and IHC staining in paraffin-embedded human lung cancer tissue (at 1:500 dilution) .

What are the optimal storage conditions for TAF4B FITC-conjugated antibodies?

For maximum stability and performance:

• Store at -20°C upon receipt

• Avoid repeated freeze/thaw cycles by making small aliquots before freezing

• Most products are supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin-300 as a preservative

• When working with the antibody, temporary storage at 4°C for short periods (1-2 weeks) is acceptable

• For long-term storage, returning aliquots to -20°C or -80°C is recommended

What is the specificity of commercially available TAF4B FITC-conjugated antibodies?

Most commercially available TAF4B FITC-conjugated antibodies:

• Are raised against specific immunogens such as recombinant Human TAF4B protein (amino acids 132-244)

• Show reactivity primarily with human TAF4B

• Are typically rabbit polyclonal antibodies with IgG isotype

• Undergo protein G purification with >95% purity

Researchers should verify cross-reactivity with their species of interest, as many antibodies are specifically validated for human samples but may work with other species based on sequence homology.

How can TAF4B FITC-conjugated antibodies be used to study its role in germ cell development?

TAF4B plays critical roles in germ cell development, particularly during prospermatogonia (ProSpg) development and quiescence. To effectively study these processes:

• Developmental time course experiments: Since TAF4B mRNA expression dramatically increases from embryonic day 12.5–18.5 , researchers can use FITC-conjugated antibodies to track protein expression changes across developmental stages.

• Co-localization studies: Combine TAF4B FITC-conjugated antibodies with markers of cell proliferation (such as PCNA) to study its role in ProSpg quiescence .

• Cell sorting approach: Use FACSAx to isolate OCT4-eGFP positive germ cells followed by immunofluorescence with TAF4B antibodies, as demonstrated in studies examining TAF4B's role in chromatin and cell cycle-related gene expression programs .

• Comparative studies: Compare TAF4B localization between wild-type and TAF4B-deficient models to identify developmental transitions where TAF4B function is crucial .

Research has shown that TAF4B-deficient ProSpg are delayed in their entry into quiescence, with altered expression of chromatin and cell cycle-related genes .

What are the considerations for using TAF4B antibodies in protein-protein interaction studies?

When studying protein-protein interactions involving TAF4B, such as the TAF4B-ZFP628 interaction , consider:

• Domain-specific targeting: The interaction between TAF4B and ZFP628 involves specific domains - amino acids 99-176 of TAF4B are necessary and amino acids 99-240 are sufficient to interact with the C-terminal 117 amino acids of ZFP628 . Choose antibodies that don't interfere with these interaction domains.

• Co-immunoprecipitation protocols: When performing co-IP experiments to validate TAF4B interactions:

  • Use testis-derived protein extracts for endogenous associations

  • Consider antibody orientation (which protein to immunoprecipitate first)

  • Validate with both forward and reverse IPs when possible

• Controls for specificity: Include appropriate controls:

  • IgG controls

  • Extracts from TAF4B-deficient tissues/cells

  • Competitive peptide blocking

• Yeast two-hybrid validation: Consider complementary Y2H assays as demonstrated in the TAF4B-ZFP628 interaction studies .

How can TAF4B FITC-conjugated antibodies be integrated with chromatin mapping techniques?

TAF4B functions in transcriptional regulation through genomic binding, which can be studied using methods like CUT&RUN (Cleavage Under Targets and Release Using Nuclease):

• Sample preparation: Isolate cells of interest (e.g., using FACS for OCT4-eGFP positive germ cells) .

• CUT&RUN protocol optimization:

  • Use H3K4me3 as a positive control for active promoters

  • Use IgG as a negative control

  • Optimize antibody concentrations (typically starting with 1:50-1:200 dilutions for TAF4B FITC antibodies)

• Data analysis approach:

  • Use tools like Homer to identify binding peaks

  • Analyze peak distribution relative to transcription start sites (TSS)

  • Integrate with RNA-seq data to correlate binding with gene expression changes

Studies have shown that TAF4B binding is primarily localized to promoter/TSS regions (73-88% of peaks), with the highest enrichment just upstream of the TSS . The integration of CUT&RUN with RNA-seq revealed that TAF4B directly regulates cell cycle and chromatin-related genes during ProSpg development .

What experimental approaches can address the tissue-specific functions of TAF4B?

TAF4B exhibits tissue-specific expression and functions, particularly in reproductive tissues. To study these specific functions:

• Cell type-specific isolation:

  • Use FACS to isolate specific cell populations (e.g., OCT4-positive germ cells)

  • Consider laser capture microdissection for tissue-specific analyses

• Comparative expression analysis:

  • Compare TAF4B expression between germ cells and somatic cells

  • Analyze expression across developmental time points

  • Study co-expression with other TFIID components

• Regulatory network mapping:

  • Investigate dependence on tissue-specific regulators like DAZL and STRA8

  • Perform motif analysis to identify co-regulatory factors (e.g., SP/KLF and NFY transcription factors)

• Functional validation in relevant models:

  • Use tissue-specific knockout models

  • Perform rescue experiments with wild-type TAF4B

Research has shown that TAF4B expression in germ cells is coordinated with other TFIID components including TAF7L and TAF9B, suggesting a specialized TFIID complex functions in germ cell development .

What are the technical challenges in using TAF4B FITC-conjugated antibodies for immunofluorescence in tissue sections?

When performing immunofluorescence with TAF4B FITC-conjugated antibodies in tissue sections, researchers should consider:

• Tissue fixation and antigen retrieval optimization:

  • For paraffin-embedded tissues, use high-pressure citrate buffer (pH 6.0) antigen retrieval as validated in lung cancer tissues

  • For frozen sections, 4% formaldehyde fixation followed by 0.2% Triton X-100 permeabilization has been successful

• Blocking considerations:

  • Use 10% normal goat serum for blocking non-specific binding

  • Consider including additional blocking steps for tissues with high autofluorescence

• Signal amplification strategies:

  • For low abundance targets, consider tyramide signal amplification

  • Use appropriate exposure settings to distinguish specific signal from background

• Multi-color imaging considerations:

  • When combining with other fluorophores, ensure minimal spectral overlap

  • Include single-color controls for spectral unmixing

• Validation approaches:

  • Use TAF4B-deficient tissues as negative controls

  • Compare staining patterns with mRNA expression data

How should researchers interpret TAF4B localization and expression in different cellular contexts?

TAF4B shows context-dependent expression and localization patterns that require careful interpretation:

• Subcellular localization:

  • TAF4B primarily localizes to the nucleus, particularly in nucleoplasm and nucleolus

  • Changes in subcellular distribution may indicate altered function

• Cell type variation:

  • Expression is highly enriched in germ cells compared to somatic cells

  • Expression increases dramatically during specific developmental transitions (e.g., E12.5-E18.5 in embryonic germ cells)

• Co-localization analysis:

  • Co-staining with other transcription factors can reveal functional associations

  • Analysis with cell cycle markers (like PCNA) can help interpret TAF4B's role in proliferation versus quiescence

• Quantification approaches:

  • Use digital image analysis for quantitative assessment of expression levels

  • Compare nuclear/cytoplasmic ratios across different cell types or conditions

• Developmental context:

  • Interpret expression patterns in light of known developmental transitions

  • Consider coordinated expression with other TFIID components

Research shows that TAF4B expression correlates with critical developmental transitions, including mitotic-to-quiescent prospermatogonia transitions .

What methodological approaches can resolve contradictory findings about TAF4B function?

Researchers studying TAF4B may encounter contradictory findings due to its context-specific functions. To address these challenges:

• Comprehensive experimental design:

  • Use multiple complementary techniques (RNA-seq, protein detection, chromatin mapping)

  • Study multiple time points to capture dynamic expression changes

  • Include both loss-of-function and gain-of-function approaches

• Control selection:

  • Include appropriate genetic controls (wild-type, heterozygous, and knockout)

  • Use multiple antibody clones targeting different epitopes

  • Validate findings with genetic approaches (CRISPR, RNAi)

• Integration of datasets:

  • Combine CUT&RUN data with RNA-seq to distinguish direct vs. indirect effects

  • Compare motif enrichment between upregulated and downregulated genes

  • Analyze protein networks to understand contextual functions

• Consideration of compensatory mechanisms:

  • Assess expression of paralogous factors (e.g., TAF4A) in TAF4B-deficient models

  • Study acute vs. chronic loss of function

Studies have revealed that TAF4B has both activating and repressive functions, with different motif enrichment patterns between genes that are upregulated versus downregulated in TAF4B-deficient cells .

How can TAF4B FITC-conjugated antibodies be used in flow cytometry applications?

For flow cytometry applications with TAF4B FITC-conjugated antibodies:

• Cell preparation protocols:

  • For intracellular staining, fix cells in 4% formaldehyde

  • Permeabilize with 0.2% Triton X-100 or commercial permeabilization buffers

  • Block with appropriate serum (e.g., 10% normal goat serum)

• Staining optimization:

  • Start with manufacturer-recommended dilutions (typically 1:50-1:200 for IF applications)

  • Perform titration experiments to determine optimal antibody concentration

  • Include single-color controls for compensation

• Gating strategies:

  • Use forward/side scatter to identify viable cells

  • Include isotype controls matched to antibody concentration

  • Consider counterstaining with DNA dyes to assess cell cycle status

• Experimental applications:

  • Sort TAF4B-positive cell populations for downstream analysis

  • Combine with cell surface markers to identify specific subpopulations

  • Quantify TAF4B expression levels across developmental stages

• Data analysis approaches:

  • Report median fluorescence intensity rather than percent positive

  • Use appropriate statistical tests for comparing expression levels

  • Consider visualization methods like UMAP for multi-parameter analysis

Flow cytometry with TAF4B antibodies can complement immunofluorescence microscopy by providing quantitative expression data across large cell populations.