PFKFB4 Antibody, FITC conjugated

What is PFKFB4 and why is it significant in cancer research?

PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4) is a bifunctional enzyme that plays a crucial role in cellular metabolic processes. It regulates glycolytic flux by controlling the levels of fructose-2,6-bisphosphate (F2,6BP), which acts as an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a key control point in the glycolytic pathway .

The significance of PFKFB4 in cancer research stems from its involvement in metabolic reprogramming, a hallmark of cancer cells. PFKFB4 expression is significantly elevated in various cancer subtypes, particularly in breast cancer compared to adjacent normal tissues . Recent studies have identified PFKFB4 in unbiased RNAi-based screens as being required for cancer cell survival . Furthermore, PFKFB4 has been shown to drive metastatic breast cancer, with elevated levels correlating with aggressive metastatic disease and reduced disease-free survival in triple-negative breast cancer (TNBC) patients .

Additionally, PFKFB4 has been implicated in drug resistance, particularly in estrogen receptor-positive breast cancer cells, where it facilitates resistance to palbociclib by reprogramming glucose metabolism and enhancing glycolysis .

What are the typical applications for PFKFB4 antibodies in experimental research?

PFKFB4 antibodies are utilized across multiple experimental applications in cancer and metabolic research:

FITC-conjugated variants are particularly valuable for direct fluorescence detection in flow cytometry, fluorescence microscopy, and high-content screening applications, eliminating the need for secondary antibody incubation steps.

How should researchers validate the specificity of PFKFB4 antibodies?

Validation of PFKFB4 antibodies, particularly FITC-conjugated versions, requires a multi-step approach:

• Positive and negative controls: Use cell lines with known PFKFB4 expression levels. According to search results, HepG2, MDA-MB-231, PC-3, and Raji cells show positive PFKFB4 expression and can serve as positive controls .

• Knockdown/knockout validation: Compare antibody signals between wild-type cells and those with PFKFB4 knockdown using siRNA or CRISPR-Cas9. The search results mention experimental approaches using doxycycline-inducible shRNAs targeting PFKFB4 that can be used for this purpose .

• Molecular weight verification: The observed molecular weight of PFKFB4 should match the calculated molecular weight of 54 kDa .

• Cross-reactivity assessment: Test the antibody on samples from different species to confirm species-specific reactivity. The available antibodies show reactivity with human and mouse samples .

• Blocking peptide competition: Preincubation with the immunizing peptide should abolish the specific signal in immunoassays.

How can PFKFB4 antibodies be used to investigate metabolic reprogramming in cancer cells?

PFKFB4 antibodies, particularly FITC-conjugated variants for fluorescence-based detection, can be instrumental in studying metabolic reprogramming in cancer through several sophisticated approaches:

• Co-localization studies: Using FITC-conjugated PFKFB4 antibodies in combination with other metabolic markers allows for the visualization of spatial relationships between PFKFB4 and other glycolytic enzymes or mitochondrial components.

• Flow cytometry analysis: Quantitative assessment of PFKFB4 expression levels across heterogeneous tumor cell populations can reveal subpopulations with distinct metabolic profiles.

• Hypoxia response investigations: PFKFB4 expression increases under hypoxic conditions, making FITC-conjugated antibodies valuable for studying adaptive metabolic responses. Research has shown that PFKFB4's requirement for glycolytic flux to lactate is significantly enhanced under hypoxic conditions .

• Metabolic flux correlation: Research has demonstrated that PFKFB4 knockdown significantly decreases steady-state fructose-2,6-bisphosphate levels, ATP concentration, and 13C-glycolytic flux to lactate and glutamate . PFKFB4 antibodies can be used alongside 13C-labeled glucose metabolic tracing to correlate enzyme expression with metabolic patterns.

• Therapeutic response monitoring: Given PFKFB4's role in palbociclib resistance in ER+ breast cancer , antibodies can monitor changes in expression patterns during treatment to predict or explain drug resistance development.

What methodological considerations are important when using FITC-conjugated PFKFB4 antibodies for immunofluorescence?

When using FITC-conjugated PFKFB4 antibodies for immunofluorescence applications, researchers should consider several important methodological factors:

• Fixation optimization: The search results indicate that for IHC applications with PFKFB4 antibodies, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may be used alternatively . Similar optimization may be required for IF applications with FITC-conjugated antibodies.

• Autofluorescence mitigation: FITC emits in the green spectrum (peak ~525 nm), which overlaps with cellular autofluorescence, particularly in certain tissues. Consider:

  • Using appropriate autofluorescence quenching reagents

  • Implementing spectral unmixing during image acquisition

  • Including unstained controls and single-color controls

• Photobleaching prevention: FITC is relatively susceptible to photobleaching. Use anti-fade mounting media and minimize exposure to light during preparation and imaging.

• Multiplexing considerations: When designing multi-color immunofluorescence experiments:

  • Pair FITC-conjugated PFKFB4 antibodies with fluorophores that have minimal spectral overlap

  • Consider the expected expression level and localization pattern of PFKFB4 relative to other target proteins

• Dilution optimization: Based on the unconjugated antibody recommendations, start with dilutions in the range of 1:200-1:800 for FITC-conjugated variants, but optimize for your specific application and antibody concentration .

How does PFKFB4 expression correlate with metastatic potential, and what techniques can assess this relationship?

Research demonstrates a significant correlation between PFKFB4 expression and metastatic potential in cancer, particularly in breast cancer. Several techniques utilizing PFKFB4 antibodies can help assess this relationship:

• Orthotopic tumor models with metastasis tracking: Studies have employed PFKFB4 knockdown in highly metastatic TNBC line LM3.3 cells implanted in mammary fat pads of SCID mice, demonstrating that PFKFB4 expression drives breast cancer recurrence and metastasis . Similar approaches using antibody-based detection of PFKFB4 can track expression dynamics during metastatic progression.

• Migration and invasion assays correlation: Boyden chamber migration and Matrigel-coated invasion assays have shown that loss of PFKFB4 robustly attenuates migratory and invasive potential of TNBC cells . FITC-conjugated PFKFB4 antibodies can quantitatively assess expression levels in these functional assays.

• Integrin pathway interaction analysis: Research has demonstrated that PFKFB4 influences cell migration toward integrin αvβ3 ligands like fibronectin and vitronectin . Co-immunoprecipitation followed by Western blotting or co-localization studies using fluorescent antibodies can reveal the molecular interactions between PFKFB4 and integrin pathway components.

• Organotropic metastasis characterization: "Early organotropic metastases" associated with elevated PFKFB4 levels have been identified in experimental models . Immunostaining of tissue samples from different metastatic sites can reveal organ-specific expression patterns.

• Patient sample correlation: Tissue microarray analysis using PFKFB4 antibodies on patient samples with known clinical outcomes can establish correlations between expression levels and metastatic burden or survival metrics.

What is the role of PFKFB4 in regulating autophagy, and how can antibodies help investigate this process?

PFKFB4 has been identified as a regulator of autophagy, primarily through its influence on cellular redox balance . FITC-conjugated PFKFB4 antibodies can facilitate the investigation of this process through several approaches:

• Co-localization with autophagy markers: Dual fluorescence labeling with FITC-conjugated PFKFB4 antibodies and markers of autophagosome formation (e.g., LC3B) can reveal spatial and temporal relationships during autophagy induction.

• Flow cytometric quantification: Measuring PFKFB4 expression levels in conjunction with autophagy indicators in response to autophagy inducers or inhibitors.

• Live-cell imaging: Using cell-permeable FITC-conjugated antibody fragments to track PFKFB4 localization dynamics during autophagy induction in real-time.

• Redox state correlation: Since PFKFB4 regulates autophagy by influencing redox balance , simultaneous assessment of PFKFB4 expression and redox indicators can illuminate this relationship.

• Glycolytic substrate manipulation: Monitoring PFKFB4 expression and localization changes when altering glucose availability or glycolytic flux, which can impact autophagy processes.

How can researchers design effective experiments to study the bifunctional enzymatic activity of PFKFB4 using antibodies?

PFKFB4 possesses both kinase and phosphatase activities with a kinase:phosphatase ratio of approximately 4.6:1 . Designing experiments to study this bifunctional nature requires careful consideration:

• Domain-specific antibodies: Consider using antibodies that target specific functional domains to distinguish between kinase and phosphatase activities. While the search results don't specify domain-specific antibodies, researchers can select antibodies targeting known catalytic regions.

• Activity assays with immunoprecipitation: Use PFKFB4 antibodies to immunoprecipitate the enzyme from cell lysates, followed by in vitro kinase or phosphatase activity assays. The enzymatic parameters established in previous research (Km for F6P = 374.2 ± 20 μM; Km for F2,6BP = 43.52 ± 5 μM) can serve as reference points.

• Metabolite level correlation: Measure F2,6BP levels in cells with varying PFKFB4 expression or under different conditions, correlating metabolite changes with antibody-detected protein levels.

• Structured illumination microscopy: High-resolution imaging using FITC-conjugated antibodies can reveal subcellular localization patterns that might correlate with differential enzymatic functions in specific cellular compartments.

• Conditional expression systems: Use inducible expression systems coupled with antibody detection to study the acute effects of PFKFB4 expression on metabolite levels and downstream glycolytic flux.

What are the optimal conditions for using PFKFB4 antibodies in Western blotting applications?

Based on the search results, the following conditions are recommended for optimal Western blotting with PFKFB4 antibodies:

For FITC-conjugated variants, additional considerations include protection from light during all steps and potential modifications to the detection system since the primary antibody is already labeled.

How can researchers troubleshoot non-specific binding or weak signals when using PFKFB4 antibodies?

When encountering issues with PFKFB4 antibodies, including FITC-conjugated versions, consider these troubleshooting approaches:

For Non-specific Binding:

• Optimize antibody concentration: Titrate the antibody to find the optimal concentration that provides specific signal with minimal background.

• Increase blocking stringency: Extend blocking time or use alternative blocking agents (BSA, normal serum, commercial blockers).

• Add detergents: Increase Tween-20 concentration in wash buffers to reduce hydrophobic interactions.

• Perform cross-adsorption: Pre-incubate the antibody with proteins from species or tissues that show cross-reactivity.

• Validate with controls: Use PFKFB4 knockout or knockdown samples to confirm specificity.

For Weak Signals:

• Optimize antigen retrieval: For IHC/ICC applications, the search results suggest using TE buffer pH 9.0 for antigen retrieval, though citrate buffer pH 6.0 is an alternative .

• Increase antibody concentration: Try a more concentrated antibody solution while monitoring background.

• Extend incubation time: Overnight incubation at 4°C often improves signal strength.

• Enhance detection sensitivity: For Western blotting, use high-sensitivity ECL substrates; for fluorescence, optimize exposure settings.

• Check sample preparation: Ensure proteins are not degraded during extraction and loading is sufficient.

How can PFKFB4 antibodies be utilized to study its role in drug resistance mechanisms?

Research has demonstrated that PFKFB4 facilitates palbociclib resistance in estrogen receptor-positive breast cancer . PFKFB4 antibodies, including FITC-conjugated variants, can be employed to investigate drug resistance mechanisms through several approaches:

• Expression monitoring during treatment: Track PFKFB4 expression levels in cancer cells before, during, and after drug treatment to identify temporal changes correlated with resistance development.

• Combination therapy assessment: Evaluate PFKFB4 expression in response to combination therapies that target both the primary cancer pathway and metabolic adaptations.

• Patient sample stratification: Use PFKFB4 antibodies to analyze patient samples and stratify responders versus non-responders to specific therapies based on expression levels.

• Metabolic pathway correlation: Research has shown that PFKFB4 reprograms glucose metabolism and promotes cell stemness by enhancing glycolysis during palbociclib resistance . Correlate PFKFB4 expression with other glycolytic markers to map the metabolic shift.

• Therapeutic targeting validation: Confirm that "diminishing PFKFB4 levels improved drug sensitivity and overcame chemoresistance during palbociclib treatment in ER+ BC" by using antibodies to validate PFKFB4 reduction in experimental models.

What considerations are important when designing multiplex immunofluorescence panels that include FITC-conjugated PFKFB4 antibodies?

When designing multiplex immunofluorescence panels incorporating FITC-conjugated PFKFB4 antibodies, researchers should consider:

• Spectral compatibility: FITC emits in the green spectrum (peak ~525 nm), so pair with fluorophores that have minimal spectral overlap, such as:

  • Far-red fluorophores (Cy5, Alexa Fluor 647)

  • Red fluorophores (Cy3, Alexa Fluor 594)

  • Blue fluorophores (DAPI for nuclear counterstaining)

• Expression level balancing: If PFKFB4 is highly expressed in your sample, consider using it with the FITC channel, which may have higher background, and reserve more sensitive fluorophores for low-abundance targets.

• Subcellular localization patterns: PFKFB4 is primarily cytoplasmic, so pair with markers in different subcellular compartments for clear distinction.

• Antibody species compatibility: When using multiple primary antibodies, they should be raised in different host species to avoid cross-reactivity with secondary antibodies.

• Sequential staining consideration: For challenging multiplex panels, consider sequential staining with stripping or quenching between rounds.

• Positive control inclusions: Include sections with known PFKFB4 expression patterns in each staining batch to ensure technical consistency.

How might PFKFB4 antibodies contribute to developing metabolic targeting strategies in cancer therapy?

PFKFB4 antibodies, including FITC-conjugated variants, can significantly contribute to developing metabolic targeting strategies for cancer therapy in several innovative ways:

• Biomarker development: PFKFB4 expression has been linked to poor outcomes in TNBC patients , suggesting potential use as a prognostic or predictive biomarker. Antibodies can help establish standardized detection protocols for clinical implementation.

• Patient stratification: Identify patient subgroups likely to benefit from glycolysis-targeting therapies based on PFKFB4 expression patterns across tumor samples.

• Therapeutic target validation: Research has shown that PFKFB4 inhibition suppresses xenograft F2,6BP levels, glucose uptake, and tumor growth in athymic mice . Antibodies can confirm target engagement and pathway modulation in preclinical models.

• Combination therapy rationale: Since PFKFB4 is involved in palbociclib resistance , antibody-based screening can identify other therapeutic agents that might synergize with PFKFB4 inhibition.

• Metabolic vulnerability mapping: Use PFKFB4 antibodies in conjunction with other metabolic markers to create comprehensive maps of cancer-specific metabolic vulnerabilities that can be therapeutically exploited.

• Tumor microenvironment interactions: Investigate how hypoxic activation of PFKFB4 in the tumor microenvironment contributes to cancer progression, potentially revealing new intervention points.

What are the methodological considerations for studying PFKFB4 in patient-derived xenograft models?

When studying PFKFB4 in patient-derived xenograft (PDX) models using antibodies, several methodological considerations are important:

• Species cross-reactivity: Select antibodies that can distinguish between human (tumor) and mouse (stroma) PFKFB4, or use species-specific antibodies. The search results indicate that many PFKFB4 antibodies are reactive with both human and mouse samples .

• Heterogeneity assessment: Use FITC-conjugated antibodies with flow cytometry to quantify heterogeneous PFKFB4 expression across different regions of PDX tumors.

• Temporal dynamics: Design longitudinal studies to track changes in PFKFB4 expression during PDX establishment and growth, which may reflect selection pressures and adaptive responses.

• Microenvironmental context: Perform co-staining with markers of hypoxia, as PFKFB4 expression and function are enhanced under hypoxic conditions .

• Treatment response correlation: Monitor PFKFB4 expression before and after therapeutic interventions in PDX models to identify patterns associated with response or resistance.

• Matched patient sample comparison: Compare PFKFB4 expression in the original patient tumor and derived PDX models to assess how faithfully the model recapitulates the metabolic phenotype.