TFDP2 Antibody, FITC conjugated

What is TFDP2 and what cellular functions does it regulate?

TFDP2 (Transcription Factor Dp-2) is a transcription cofactor that binds DNA cooperatively with E2F family members through the E2 recognition site (5′-TTTC[CG]CGC-3′) found in promoter regions of genes involved in cell cycle regulation and DNA replication. The TFDP2:E2F complex functions critically in controlling cell-cycle progression from G1 to S phase. Additionally, the E2F1:DP complex appears to mediate both cell proliferation and apoptosis. TFDP2 also blocks adipocyte differentiation by repressing CEBPA binding to its target gene promoters .

In erythroid cells, TFDP2 is essential for proper erythroid differentiation, with its expression being highly upregulated during terminal erythropoiesis. TFDP2 knockdown leads to significantly reduced rates of proliferation and reduced upregulation of many erythroid-important genes, including alpha and beta hemoglobin chains (Hbb-b1 and Hba-a1), GATA1, and enzymes required for heme biosynthesis .

What are the key specifications of commercially available TFDP2 Antibody, FITC conjugated?

The FITC-conjugated TFDP2 antibody is typically a polyclonal antibody raised in rabbits against human TFDP2. The specifications include:

Sources:

How should I store and handle FITC-conjugated TFDP2 antibody to maintain its activity?

The FITC-conjugated TFDP2 antibody should be stored according to these guidelines to maintain optimal activity:

• Upon receipt, aliquot the antibody to avoid repeated freeze-thaw cycles

• Store at -20°C or -80°C in a freezer (not frost-free to avoid freeze-thaw cycles)

• Protect from light exposure as FITC is light-sensitive

• When thawing for use, thaw quickly at room temperature and keep on ice once thawed

• Return unused portion to -20°C immediately after use

• Avoid exposure to high temperatures or extreme pH conditions

• For long-term storage stability, maintain in the buffer provided (PBS, pH 7.3-7.4 with 0.03% Proclin 300 and 50% glycerol)

What are the optimal conditions for using FITC-conjugated TFDP2 antibody in fluorescence microscopy?

For optimal fluorescence microscopy using FITC-conjugated TFDP2 antibody:

• Fixation protocol:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Alternatively, use methanol fixation (-20°C for 10 minutes) for nuclear proteins like TFDP2

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to allow antibody access to nuclear proteins

• Blocking:

  • Block with 5% normal serum (from the same species as the secondary antibody if used) in PBS with 0.1% Tween-20 for 1 hour at room temperature

• Antibody dilution:

  • Start with a 1:50 to 1:200 dilution range in blocking buffer

  • Optimize through titration experiments for your specific cell type

• Incubation conditions:

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • For co-localization studies with E2F proteins, ensure minimal spectral overlap with other fluorophores

• Counterstaining:

  • Use DAPI (1 μg/ml) for nuclear visualization

  • Mount in anti-fade mounting medium to preserve FITC fluorescence

• Microscopy settings:

  • Excitation maximum: 495 nm

  • Emission maximum: 519 nm

  • Use appropriate filter sets to minimize photobleaching

How can I optimize Western blot protocols when using TFDP2 antibodies?

While the FITC-conjugated antibody is primarily designed for immunofluorescence applications, TFDP2 antibodies can be used in Western blot with these optimization strategies:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell lysis

  • For nuclear proteins like TFDP2, consider using nuclear extraction protocols

  • Denature samples at 95°C for 5 minutes in Laemmli buffer with DTT

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels (TFDP2 observed MW: 50 kDa)

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • For non-conjugated TFDP2 antibodies, dilute 1:200 to 1:2000 in blocking solution

  • Incubate overnight at 4°C with gentle rocking

• Detection considerations:

  • For unconjugated antibodies, use HRP-conjugated secondary antibodies

  • If using pre-conjugated antibodies, ensure the fluorophore/enzyme is compatible with your detection system

  • Expected band size: 50 kDa (observed); multiple bands may be detected (34-49 kDa) due to different isoforms

• Validation controls:

  • Include positive control lysates from cells known to express TFDP2

  • Consider using TFDP2 knockdown samples as negative controls

What are the recommended protocols for co-immunoprecipitation of TFDP2 with E2F partners?

Since TFDP2 functions through interaction with E2F transcription factors, co-immunoprecipitation (Co-IP) is valuable for studying these interactions:

• Lysis buffer composition:

  • 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate

  • Include protease inhibitors, phosphatase inhibitors, and 1 mM DTT

  • For nuclear proteins, add 10% glycerol and 0.1% SDS

• Pre-clearing step:

  • Incubate lysates with Protein G beads for 1 hour at 4°C

  • Remove beads to reduce non-specific binding

• Immunoprecipitation:

  • Add 2-5 μg of anti-TFDP2 antibody to 500 μg of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add Protein G beads for 2-4 hours at 4°C

  • Wash 4-5 times with wash buffer (lysis buffer with reduced detergent)

• Elution and analysis:

  • Elute with 2X Laemmli buffer at 95°C for 5 minutes

  • Analyze by Western blot using antibodies against E2F family members (especially E2F2)

  • Reverse Co-IP using E2F antibodies can validate interactions

• Controls:

  • IgG control: Use the same amount of non-specific IgG from the same species

  • Input sample: Load 5-10% of pre-immunoprecipitation lysate

  • Negative control: Use cells with TFDP2 knockdown

Given that the TFDP2:E2F complex functions in the control of cell-cycle progression from G1 to S phase, this protocol is particularly useful for studying how these interactions change throughout the cell cycle .

How does TFDP2 contribute to erythroid differentiation and what methods can assess this function?

TFDP2 plays a critical role in erythropoiesis through several mechanisms:

• Transcriptional regulation: TFDP2 and its partner E2F2 are highly induced during terminal erythropoiesis, with expression patterns showing the greatest induction occurring at the R2 to R3 transition of erythroid maturation .

• Regulation by master erythroid transcription factors: GATA1 and TAL1 bind to regulatory regions of the Tfdp2 gene to upregulate its expression. ChIP-seq data identified two regions of co-occupancy: one in the putative promoter region 5′ of the transcriptional start site (H3K4me3-positive and H3K4me1-negative) and another in the first intron of Tfdp2 .

• Functional impact: Knockdown of Tfdp2 results in:

  • Significantly reduced rates of proliferation

  • Reduced upregulation of erythroid-important genes

  • Global inhibition of normal downregulation of E2F2 target genes

  • Cell cycle disruption with accumulation in S phase

  • Increased erythrocyte size

  • Impaired hemoglobin synthesis

Methods to assess TFDP2 function in erythropoiesis include:

• shRNA-mediated knockdown: Using vectors like MSCV-pkgGFP-U3-U6P with shRNA sequences targeting Tfdp2

• Gene expression analysis: RT-qPCR to measure levels of erythroid markers (Hbb-b1, Hba-a1, GATA1, Epb4.1)

• Cell cycle analysis: Flow cytometry to assess cell cycle distribution

• Luciferase reporter assays: To evaluate GATA1/TAL1-mediated activation of Tfdp2 regulatory regions

• ChIP-seq: To identify TFDP2 binding sites genome-wide

• Cell morphology assessment: To evaluate changes in erythrocyte size and shape

What are the technical considerations for using TFDP2 antibodies in ChIP-seq experiments?

Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) is valuable for mapping TFDP2 binding sites genome-wide. Key technical considerations include:

• Cross-linking conditions:

  • Standard: 1% formaldehyde for 10 minutes at room temperature

  • For indirect DNA binding proteins like TFDP2, consider dual cross-linking with 1 mM DSG (disuccinimidyl glutarate) for 30 minutes followed by formaldehyde

• Sonication parameters:

  • Target fragment size: 200-300 bp

  • Optimize sonication time and amplitude for your specific cell type

  • Verify fragment size by agarose gel electrophoresis

• Antibody selection and validation:

  • Use ChIP-grade antibodies if available

  • For FITC-conjugated antibodies, ensure the conjugation doesn't interfere with epitope recognition

  • Validate antibody specificity by Western blot and immunoprecipitation

  • Include IgG controls and input samples

• Immunoprecipitation conditions:

  • Use 2-5 μg antibody per ChIP reaction

  • Include negative controls (IgG) and positive controls (known targets)

  • Consider including TFDP2 knockdown samples as specificity controls

• Analysis considerations:

  • Look for co-occupancy with E2F family members

  • Cross-reference with GATA1 and TAL1 binding sites in erythroid cells

  • Analyze for E2F recognition motifs (5′-TTTC[CG]CGC-3′)

  • Integrate with gene expression data to identify functional targets

• Technical challenges:

  • As TFDP2 binds DNA indirectly through E2F proteins, signal-to-noise ratio may be lower

  • Consider sequential ChIP (Re-ChIP) to enrich for TFDP2-E2F co-occupied regions

How can TFDP2 antibodies be used to investigate cell cycle-dependent regulation and function?

TFDP2 and its E2F partners are critical regulators of cell cycle progression. Here are methodological approaches to investigate cell cycle-dependent regulation and function:

• Cell synchronization techniques:

  • Double thymidine block: Synchronize cells at G1/S boundary

  • Nocodazole treatment: Synchronize cells in M phase

  • Serum starvation/stimulation: Synchronize cells in G0/G1

• Flow cytometry analysis:

  • Combine FITC-conjugated TFDP2 antibody with DNA content staining (propidium iodide or DAPI)

  • Analyze TFDP2 expression levels across cell cycle phases

  • Consider dual staining with cyclin markers for precise cell cycle staging

• Live-cell imaging approaches:

  • Use FITC-conjugated TFDP2 antibodies with cell-permeable delivery methods

  • Combine with fluorescently tagged cell cycle markers

  • Monitor real-time changes in TFDP2 localization and expression

• Functional genomics approaches:

  • Perform TFDP2 ChIP-seq at defined cell cycle stages

  • Integrate with RNA-seq data to correlate binding with transcriptional outcomes

  • Compare TFDP2 binding patterns across G1, S, G2, and M phases

• Proximity ligation assays (PLA):

  • Detect TFDP2-E2F interactions in situ

  • Quantify changes in interaction frequency across cell cycle phases

  • Combine with cell cycle markers for precise staging

• Phosphorylation-specific analyses:

  • Investigate cell cycle-dependent post-translational modifications of TFDP2

  • Use phospho-specific antibodies if available

  • Consider mass spectrometry approaches to identify novel modifications

Since loss of TFDP2 causes cells to accumulate in S phase and results in increased erythrocyte size, these methodologies can help elucidate the mechanisms by which TFDP2 couples cell cycle progression with differentiation .

What are common issues when using FITC-conjugated antibodies and how can they be addressed?

Researchers frequently encounter these issues when working with FITC-conjugated antibodies like TFDP2:

• Photobleaching:

  • Problem: FITC is particularly susceptible to photobleaching

  • Solutions:

    • Add anti-fade reagents to mounting medium

    • Minimize exposure to excitation light during imaging

    • Consider using Tyramide Signal Amplification (TSA) for signal enhancement

    • Store slides in the dark at 4°C

• High background fluorescence:

  • Problem: Non-specific binding or autofluorescence

  • Solutions:

    • Optimize blocking (try different serums or BSA concentrations)

    • Increase washing steps (duration and number)

    • Include 0.1-0.3% Triton X-100 in washing buffer

    • Use Sudan Black B (0.1-0.3%) to reduce autofluorescence

    • Test different fixatives (paraformaldehyde vs. methanol)

• Weak or no signal:

  • Problem: Insufficient antibody concentration or epitope masking

  • Solutions:

    • Titrate antibody concentration (try 1:50 to 1:500 dilutions)

    • Optimize antigen retrieval methods for fixed tissues

    • Extend primary antibody incubation time (overnight at 4°C)

    • Check pH of buffers (FITC performs best at slightly alkaline pH)

• Cross-reactivity:

  • Problem: Non-specific binding to unintended targets

  • Solutions:

    • Pre-absorb antibody with related proteins

    • Include additional blocking steps

    • Validate with knockout/knockdown controls

    • Use more stringent washing conditions

• pH sensitivity:

  • Problem: FITC fluorescence decreases at pH < 7.0

  • Solutions:

    • Ensure all buffers are at pH 7.2-8.0

    • Check pH of mounting medium

    • Avoid acidic fixatives

How can I validate the specificity of TFDP2 antibody staining in my experimental system?

Validating antibody specificity is crucial for reliable results. Here are methodological approaches for TFDP2 antibody validation:

• Genetic approaches:

  • siRNA/shRNA knockdown: Compare staining in control vs. TFDP2-depleted cells

  • CRISPR/Cas9 knockout: Generate TFDP2-null cells as negative controls

  • Overexpression: Analyze cells overexpressing tagged TFDP2 for co-localization

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (if available)

  • Compare staining with and without peptide competition

  • Specific signals should be significantly reduced after competition

• Multiple antibody validation:

  • Compare staining patterns using antibodies targeting different TFDP2 epitopes

  • Consistent patterns across antibodies suggest specific detection

• Western blot correlation:

  • Verify that cell types showing strong immunofluorescence also show strong bands on Western blot

  • Compare relative expression levels across techniques

• Subcellular localization assessment:

  • TFDP2 is primarily nuclear; confirm nuclear localization with DAPI counterstaining

  • Aberrant staining patterns (e.g., strong cytoplasmic signal) may indicate non-specificity

• Cell cycle phase-specific validation:

  • TFDP2 expression varies through the cell cycle

  • Correlate staining intensity with cell cycle markers or synchronized populations

  • Expression typically increases during G1/S transition

What strategies can address discrepancies between antibody-based detection and mRNA expression data for TFDP2?

Researchers sometimes observe discrepancies between TFDP2 protein levels (detected by antibodies) and mRNA expression. Consider these methodological approaches to investigate and address such discrepancies:

• Post-transcriptional regulation assessment:

  • Analyze miRNA targeting of TFDP2 (miR-93 and miR-20b have been reported to target TFDP2)

  • Measure TFDP2 mRNA stability through actinomycin D chase experiments

  • Investigate alternative splicing using isoform-specific primers

• Post-translational modification analysis:

  • Assess protein stability using cycloheximide chase experiments

  • Investigate ubiquitination status and proteasome-dependent degradation

  • Analyze phosphorylation states that might affect antibody recognition

• Technical validation:

  • Test antibody recognition across multiple TFDP2 isoforms (calculated MW ranges from 34-49 kDa)

  • Verify epitope accessibility in different experimental conditions

  • Ensure primer specificity for RT-qPCR detection of all relevant isoforms

• Temporal dynamics investigation:

  • Perform time-course analyses since mRNA and protein levels may be temporally offset

  • Monitor both mRNA and protein during cell cycle progression or differentiation

• Single-cell analysis approaches:

  • Combine immunofluorescence with RNA-FISH to correlate mRNA and protein levels at single-cell resolution

  • Assess cell-to-cell variability that might be masked in population-level analyses

• Experimental validation strategies:

  • Express TFDP2 from an exogenous promoter and measure protein accumulation

  • Test effects of proteasome inhibitors on TFDP2 protein levels

  • Analyze polysome association of TFDP2 mRNA to assess translation efficiency

How can TFDP2 antibodies be used to investigate its role in diseases beyond erythropoiesis?

TFDP2 functions in cell cycle regulation and differentiation suggest potential roles in various pathological conditions. Here are methodological approaches to investigate these roles:

• Cancer research applications:

  • Immunohistochemistry/immunofluorescence on tissue microarrays to assess expression across tumor types

  • Correlation of TFDP2 levels with clinical outcomes and cancer subtypes

  • Analysis of TFDP2:E2F complexes in tumor samples using proximity ligation assays

  • Investigation of TFDP2 in therapy resistance mechanisms through knockdown/overexpression studies

• Developmental disorders:

  • Immunostaining of developmental tissue series to track TFDP2 expression patterns

  • Analysis of patient-derived cells harboring mutations in cell cycle regulators

  • Investigation of TFDP2 in stem cell differentiation models

• Metabolic disease models:

  • Given TFDP2's role in blocking adipocyte differentiation, analyze expression in adipose tissue from metabolic disease models

  • Investigate TFDP2-CEBPA interactions in hepatic and adipose tissues

  • Correlate TFDP2 levels with insulin signaling pathway components

• Inflammation and immune response:

  • Analyze TFDP2 dynamics during immune cell activation and proliferation

  • Investigate potential roles in cytokine-induced cell cycle entry

  • Study TFDP2 in models of inflammatory diseases

• Neurodegenerative conditions:

  • Assess TFDP2 in post-mitotic neurons under stress conditions

  • Investigate potential re-entry into cell cycle as a pathological mechanism

  • Analyze TFDP2:E2F complexes in neurodegenerative disease models

What are the considerations for multiplexed imaging approaches involving FITC-conjugated TFDP2 antibodies?

Multiplexed imaging allows simultaneous detection of TFDP2 with other proteins of interest. Technical considerations include:

• Spectral compatibility planning:

  • FITC excitation/emission: 495/519 nm

  • Choose fluorophores with minimal spectral overlap (recommended partners: Cy3, Cy5, APC)

  • Consider spectral unmixing algorithms for closely overlapping fluorophores

• Sequential staining protocols:

  • For antibodies raised in the same species, use sequential staining with intermediate blocking

  • Consider tyramide signal amplification (TSA) for sequential same-species antibody use

  • Test order of antibody application to optimize signal intensity

• Controls for multiplexed imaging:

  • Single-stain controls for spectral compensation

  • Fluorescence-minus-one (FMO) controls to set gating thresholds

  • Isotype controls for each fluorophore

• Protein partners for co-localization studies:

  • E2F family members (particularly E2F2) to study functional complexes

  • Cell cycle markers (cyclins, CDKs) to correlate with cell cycle phase

  • Transcriptional machinery components to study active transcription sites

  • GATA1 and TAL1 in erythroid cells to study regulatory relationships

• Multiplexing technologies:

  • Consider cyclic immunofluorescence for high-dimensional imaging

  • Mass cytometry (CyTOF) using metal-tagged antibodies for higher multiplexing

  • Imaging mass cytometry for tissue section analysis

  • DNA-barcoded antibodies for highly multiplexed detection

How can TFDP2 antibodies be integrated into single-cell analysis workflows?

Integrating TFDP2 antibody-based detection into single-cell analysis provides insights into heterogeneity of expression and function. Methodological considerations include:

• Single-cell protein detection methods:

  • Flow cytometry: Combine FITC-TFDP2 antibody with cell cycle markers and other proteins of interest

  • Mass cytometry (CyTOF): Use metal-tagged TFDP2 antibodies for high-parameter analysis

  • CITE-seq: Combine antibody detection with single-cell RNA-seq using DNA-barcoded antibodies

• Spatial transcriptomics integration:

  • Combine TFDP2 immunofluorescence with in situ RNA detection methods

  • Correlate protein localization with transcriptome data at single-cell resolution

  • Use sequential immunofluorescence and RNA-FISH for multi-parameter analysis

• Live-cell imaging applications:

  • Use cell-permeable fluorescent nanobodies against TFDP2 if available

  • Combine with fluorescent cell cycle reporters (FUCCI system)

  • Track TFDP2 dynamics during differentiation or cell cycle progression

• Single-cell multi-omics approaches:

  • REAP-seq or CITE-seq: Protein and RNA detection in the same cells

  • Integrate with single-cell ATAC-seq data to correlate chromatin accessibility with TFDP2 expression

  • Correlate post-translational modifications with transcriptional states

• Computational analysis considerations:

  • Clustering algorithms to identify cell subpopulations based on TFDP2 and other markers

  • Trajectory analysis to map TFDP2 dynamics during differentiation

  • Network analysis to infer TFDP2 regulatory relationships at single-cell resolution

• Technical validation approaches:

  • Spike-in controls for antibody staining quantification

  • Batch correction methods for integrating multiple experiments

  • Signal normalization approaches to account for technical variability