PFKFB4 Antibody

What is PFKFB4 and what is its functional significance in cellular metabolism?

PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) is a bi-functional enzyme that plays a crucial role in glycolytic regulation. It possesses two enzymatic activities: as a kinase, it synthesizes fructose 2,6-bisphosphate (F2,6-BP) to stimulate glycolysis; as a phosphatase, it hydrolyzes F2,6-BP into fructose-6-phosphate (F6P) and inorganic phosphate (Pi) . PFKFB4 also functions as a protein kinase to phosphorylate various proteins, extending its regulatory influence beyond direct metabolic control .

The enzyme's significance lies in its control over F2,6-BP levels, which serves as a potent allosteric activator of 6-phosphofructo-1-kinase, a rate-limiting enzyme in glycolysis . This regulatory function is particularly important in cancer cells, which often exhibit the Warburg effect - a preference for glycolysis even in oxygen-rich conditions. Research has demonstrated elevated PFKFB4 expression in multiple cancer types, including prostate and lung cancer, suggesting its crucial role in supporting tumor growth through metabolic reprogramming .

In prostate cancer research, PFKFB4 has been implicated in androgen-independent growth, with studies showing increased glucose consumption correlating with PFKFB4 upregulation during the transition to castration resistance . This metabolic adaptation mechanism highlights PFKFB4's potential as both a biomarker and therapeutic target in advanced cancers.

What types of PFKFB4 antibodies are available and how should researchers select the appropriate one?

Researchers have access to several types of PFKFB4 antibodies, each optimized for specific applications and experimental systems:

• Based on production method:

  • Polyclonal antibodies: Most commercially available PFKFB4 antibodies are polyclonal, including Proteintech's 29902-1-AP (rabbit polyclonal IgG) and Assay Genie's PACO01324 . These recognize multiple epitopes on the PFKFB4 protein, potentially offering greater sensitivity.

• Based on species reactivity:

  • Human-specific antibodies: Some antibodies are optimized for human PFKFB4 detection only

  • Multi-species reactive antibodies: Proteintech's 29902-1-AP shows reactivity with both human and mouse samples, while Assay Genie's PACO01324 has been validated for human, mouse, and rat samples .

Selection criteria should include:

• Intended application: Verify the antibody has been validated for your specific application (WB, IHC, IF/ICC)

• Target species: Ensure compatibility with your experimental model

• Epitope location: Consider whether your experiment requires detection of specific domains

• Validation data: Review manufacturer-provided validation in relevant cell lines and tissues

• Literature citations: Prioritize antibodies with published validation in your application

Proteintech's antibody has been tested in multiple cell lines, including HepG2, MDA-MB-231, PC-3, and Raji cells for Western blotting applications, and in human kidney and mouse testis tissues for immunohistochemistry . This extensive validation across diverse biological samples provides confidence in experimental reproducibility.

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

For optimal Western blotting results with PFKFB4 antibodies, researchers should consider the following methodological parameters:

• Antibody dilution: The recommended dilution ranges vary by manufacturer:

  • Proteintech's 29902-1-AP: 1:1000-1:4000

  • Assay Genie's PACO01324: 1:500-1:2000

• Sample preparation:

  • PFKFB4 has a calculated and observed molecular weight of 54 kDa

  • Standard RIPA or NP-40 lysis buffers are generally suitable for extraction

  • Include protease inhibitors to prevent degradation

  • For phosphorylation studies, include phosphatase inhibitors

• Positive controls:

  • Cell lines with confirmed PFKFB4 expression include HepG2, MDA-MB-231, PC-3, and Raji cells

  • Consider including a PFKFB4-overexpressing lysate as a positive control

• Blocking conditions:

  • 5% non-fat dry milk or BSA in TBST is typically effective

  • For phospho-specific detection, BSA is preferred over milk

• Detection system:

  • Both chemiluminescence and fluorescence-based systems are compatible

  • For quantitative analysis, fluorescence-based detection offers better linearity

• Optimization considerations:

  • Perform a dilution series when first using the antibody with your samples

  • The optimal dilution may vary depending on expression levels in your specific samples

  • Longer exposure times may be needed for tissues with lower PFKFB4 expression

When troubleshooting, researchers should verify the expected molecular weight (54 kDa) and consider the possibility of post-translational modifications that might affect migration patterns. Sample-dependent optimization is recommended to obtain optimal results in each experimental system .

What considerations should be made when using PFKFB4 antibodies for immunohistochemistry in tissue samples?

When employing PFKFB4 antibodies for immunohistochemistry, researchers should address several critical methodological considerations:

• Tissue preparation and fixation:

  • Standard formalin fixation and paraffin embedding protocols are generally suitable

  • Optimization of fixation time may be necessary to prevent epitope masking

• Antigen retrieval:

  • For Proteintech's 29902-1-AP antibody, TE buffer at pH 9.0 is the recommended primary method

  • Alternatively, citrate buffer at pH 6.0 can be used if TE buffer yields suboptimal results

  • Heat-induced epitope retrieval (pressure cooker or microwave) is typically more effective than enzymatic methods

• Antibody dilution and incubation:

  • Recommended dilution range: 1:50-1:500

  • Optimal dilution should be determined empirically for each tissue type

  • Incubation at 4°C overnight often yields better results than shorter incubations at room temperature

• Detection systems:

  • DAB (3,3'-diaminobenzidine) is the most commonly used chromogen

  • Amplification systems may improve sensitivity for low-expression samples

• Validated positive controls:

  • Human kidney tissue and mouse testis tissue have been confirmed as positive controls

  • Cancer tissues, particularly prostate and lung cancers, typically show strong PFKFB4 expression

• Scoring and interpretation:

  • In published studies, PFKFB4 immunostaining has been categorized into four grades based on staining intensity

  • For statistical analysis, results are often dichotomized as positive or negative

• Comparative analysis considerations:

  • Studies have shown significant differences in PFKFB4 expression between:

    • Prostate cancer vs. benign prostatic hyperplasia (p < 0.000)

    • Prostate cancer vs. adjacent normal tissue (p < 0.001)

This table summarizes published PFKFB4 expression patterns across different tissue types:

These methodological details are essential for reliable PFKFB4 immunohistochemical analysis in both research and potential clinical applications.

How should PFKFB4 antibodies be properly stored and handled to maintain optimal reactivity?

Proper storage and handling of PFKFB4 antibodies is crucial for maintaining their specificity and sensitivity over time. Researchers should follow these evidence-based recommendations:

Following these storage and handling guidelines will help ensure consistent experimental results and maximize the useful life of PFKFB4 antibodies in research applications.

How can PFKFB4 antibodies be used to investigate the relationship between metabolic reprogramming and cancer progression?

PFKFB4 antibodies serve as powerful tools for exploring the connection between metabolic reprogramming and cancer progression through multiple experimental approaches:

• Comparative expression analysis:

  • Immunohistochemical staining of tissue microarrays reveals PFKFB4 upregulation across cancer types

  • Studies have demonstrated significantly higher PFKFB4 expression in prostate tumors compared to benign prostatic hyperplasia (p < 0.000)

  • Similarly, lung cancer tissues show elevated PFKFB4 expression compared to matched normal controls

  • These patterns support PFKFB4's role in cancer-specific metabolic adaptation

• Correlation with disease progression:

  • PFKFB4 has been identified as a promising biomarker for poor prognosis in gastric cancer

  • In prostate cancer, PFKFB4 expression increases during the transition from androgen-dependent to androgen-independent states

  • This temporal relationship suggests PFKFB4 may drive metabolic adaptations supporting therapy resistance

• Functional metabolic analyses:

  • Combining PFKFB4 immunoblotting with glucose consumption and lactate production measurements

  • Research protocols have established methods to correlate PFKFB4 expression with the Warburg effect in cancer models

  • This approach allows researchers to directly link PFKFB4 levels to glycolytic flux alterations

• Mechanistic investigations:

  • Co-immunoprecipitation experiments using PFKFB4 antibodies have revealed novel protein interactions

  • Mass spectrometry analysis of immunoprecipitated PFKFB4 identified FBXO28 as an interaction partner

  • This discovery uncovered PFKFB4's role in regulating HIF-1α ubiquitylation and degradation

  • The finding demonstrates how PFKFB4 connects metabolic regulation with hypoxia signaling in cancer

• Therapeutic target validation:

  • PFKFB4 antibodies enable monitoring of target engagement in drug development

  • The cancer-specific expression pattern of PFKFB4 makes it an attractive therapeutic target

  • Antibody-based detection can validate PFKFB4 inhibition in preclinical models

These applications of PFKFB4 antibodies have contributed to our understanding of how metabolic reprogramming supports cancer hallmarks including sustained proliferation, resistance to cell death, and adaptation to hypoxia. The evidence positions PFKFB4 at the intersection of metabolism and oncogenic signaling networks.

What methodological approaches can be used to validate PFKFB4 antibody specificity in experimental systems?

Rigorous validation of PFKFB4 antibody specificity is essential for generating reliable research data. Researchers should implement multiple complementary approaches:

• Genetic knockdown/knockout validation:

  • Compare antibody signal in wild-type cells versus PFKFB4-depleted samples (siRNA, shRNA, or CRISPR)

  • A specific antibody should show significantly reduced signal in PFKFB4-depleted samples

  • This approach provides the strongest functional validation of specificity

• Overexpression systems:

  • Compare detection in cells with endogenous versus overexpressed PFKFB4

  • Verify increased signal intensity corresponding to increased expression levels

  • Tag-based detection (FLAG, HA, etc.) can provide independent confirmation

• Molecular weight verification:

  • PFKFB4 has a calculated and observed molecular weight of 54 kDa

  • Verify that the primary band detected corresponds to this expected size

  • Be aware that post-translational modifications might alter apparent molecular weight

• Cross-reactivity assessment:

  • Test the antibody against related PFKFB family members (PFKFB1-3)

  • Important especially for antibodies claiming to detect multiple isoforms, like the PACO01324 antibody that detects both PFKFB1 and PFKFB4

• Multi-application concordance:

  • Compare PFKFB4 detection across different techniques (WB, IHC, IF)

  • Consistent localization and expression patterns across methods increases confidence in specificity

• Tissue-specific expression profiles:

  • Verify detection patterns match known PFKFB4 expression profiles

  • Positive controls should include tissues known to express PFKFB4:

    • Human kidney tissue

    • Mouse testis tissue

    • Cancer cell lines: HepG2, MDA-MB-231, PC-3, Raji cells

• Peptide competition assays:

  • Pre-incubate antibody with the immunogen peptide prior to application

  • Should observe significant signal reduction with peptide competition

  • Particularly useful for polyclonal antibodies like 29902-1-AP and PACO01324

• Mass spectrometry confirmation:

  • Immunoprecipitate with PFKFB4 antibody followed by mass spectrometry analysis

  • Confirms the identity of the detected protein and reveals potential cross-reactivity

Documentation of these validation steps is essential for ensuring experimental reproducibility and data reliability. Manufacturers like Proteintech provide validation data showing PFKFB4 detection in specific cell lines and tissues, which serves as a baseline for researchers' own validation experiments .

How does PFKFB4 contribute to the Warburg effect in cancer cells and how can this be studied?

PFKFB4 plays a critical role in facilitating the Warburg effect - the preference of cancer cells for glycolysis even in oxygen-rich conditions. Researchers can investigate this relationship using various methodological approaches:

• Mechanistic role of PFKFB4 in glycolytic regulation:

  • PFKFB4 controls the synthesis and degradation of fructose-2,6-bisphosphate (F2,6-BP), a potent allosteric activator of phosphofructokinase-1 (PFK-1)

  • PFK-1 catalyzes a rate-limiting step in glycolysis, converting fructose-6-phosphate to fructose-1,6-bisphosphate

  • By modulating F2,6-BP levels, PFKFB4 directly influences glycolytic flux

  • Its bifunctional nature allows context-specific metabolic adaptation

• Experimental approaches to study PFKFB4's role in the Warburg effect:

  a. Metabolic flux analysis:

  • Measure glucose consumption and lactate production in cells with PFKFB4 manipulation

  • Research protocols have established methods to correlate PFKFB4 expression with glycolytic parameters

  • Example methodology: Culture cells in medium containing standardized glucose concentrations, then measure remaining glucose and produced lactate in collected supernatants

  b. Expression correlation studies:

  • Analyze PFKFB4 expression across normal versus cancer tissues

  • Studies have demonstrated significantly higher PFKFB4 expression in:

    • Prostate tumors compared to benign prostatic hyperplasia (p < 0.000)

    • Prostate cancer compared to adjacent normal tissue (p < 0.001)

  • These patterns support PFKFB4's role in cancer-specific metabolic reprogramming

  c. Therapeutic targeting experiments:

  • Investigate metabolic consequences of PFKFB4 inhibition in cancer models

  • Monitor changes in glycolytic parameters upon PFKFB4 depletion

  • Assess impact on cancer cell proliferation and survival

• Clinical relevance of PFKFB4-driven metabolic reprogramming:

  • PFKFB4 expression increases during therapy resistance development

  • In prostate cancer, elevated PFKFB4 levels correlate with the transition to androgen independence

  • This suggests PFKFB4-mediated metabolic adaptations may support therapy resistance mechanisms

• Interaction with hypoxia pathways:

  • PFKFB4 silencing in glioma stem cells leads to downregulation of hypoxia-related genes and reduction in HIF protein levels

  • This connection between PFKFB4 and HIF signaling represents an important intersection between metabolic regulation and hypoxia adaptation

  • The relationship can be studied through co-immunoprecipitation and proteomic analysis approaches

Through these methodological approaches, researchers can dissect the multifaceted role of PFKFB4 in promoting and maintaining the Warburg effect in cancer cells, potentially identifying new therapeutic vulnerabilities.

What is the relationship between PFKFB4 and HIF-1α signaling in cancer, and how can antibody-based approaches investigate this connection?

The relationship between PFKFB4 and HIF-1α represents a critical intersection between metabolic regulation and hypoxia signaling in cancer. Antibody-based approaches offer powerful tools to investigate this connection:

• Regulatory relationship:

  • Research has revealed that PFKFB4 regulates the ubiquitylation and subsequent proteasomal degradation of HIF-1α

  • This regulation is mediated through PFKFB4's interaction with FBXO28, an F-box protein component of an E3 ubiquitin ligase complex

  • PFKFB4 silencing in glioma stem cells results in downregulation of hypoxia-related genes and dramatic reduction of HIF protein levels

  • This represents a non-metabolic function of PFKFB4 in cancer biology

• Antibody-based methodological approaches:

  a. Co-immunoprecipitation (Co-IP):

  • Use PFKFB4 antibodies to pull down associated proteins from cell lysates

  • Western blot analysis of precipitated material using HIF-1α and FBXO28 antibodies

  • Reverse Co-IP using HIF-1α or FBXO28 antibodies with PFKFB4 detection

  • This approach has successfully identified FBXO28 as a PFKFB4 interaction partner

  b. Proximity ligation assay (PLA):

  • Combine PFKFB4 and HIF-1α/FBXO28 antibodies to visualize protein-protein interactions in situ

  • Provides spatial information about where interactions occur within cells

  • Allows quantification of interaction events

  c. Immunofluorescence co-localization:

  • Simultaneous detection of PFKFB4 with HIF-1α or FBXO28

  • Analyze subcellular co-localization patterns under different conditions

  • Especially informative under hypoxic versus normoxic conditions

  d. Chromatin immunoprecipitation (ChIP):

  • Use HIF-1α antibodies for ChIP followed by qPCR for HIF target genes

  • Compare HIF-1α binding in control versus PFKFB4-depleted cells

  • Reveals functional impact of PFKFB4 on HIF-1α transcriptional activity

• Ubiquitination assays:

  • Immunoprecipitate HIF-1α under denaturing conditions

  • Probe for ubiquitin modifications by Western blot

  • Compare ubiquitination patterns in control versus PFKFB4-manipulated cells

  • This approach has demonstrated PFKFB4's role in regulating HIF-1α ubiquitylation

• Therapeutic implications:

  • The discovery of PFKFB4's role in HIF-1α regulation provides a potential new strategy for inhibiting HIF-1α in cancer cells

  • Given PFKFB4's cancer specificity, targeting this pathway might offer more selective HIF-1α inhibition compared to direct HIF inhibitors

  • Antibody-based detection methods can validate target engagement in drug development efforts

These methodological approaches have revealed that PFKFB4 functions beyond its canonical metabolic role, highlighting how antibody-based techniques continue to uncover novel protein functions and potential therapeutic targets in cancer biology.

How can researchers optimize PFKFB4 antibody-based immunoprecipitation for protein interaction studies?

Optimizing PFKFB4 antibody-based immunoprecipitation (IP) is crucial for successful protein interaction studies, particularly when investigating novel binding partners such as FBXO28 . Researchers should consider these methodological refinements:

• Antibody selection for IP:

  • Not all PFKFB4 antibodies are equally effective for immunoprecipitation

  • Verify the antibody has been validated for IP applications

  • Consider epitope location - antibodies targeting regions involved in protein interactions may disrupt those interactions

• Cell lysis optimization:

  • Buffer composition critically affects protein complex preservation:

    • NP-40 or Triton X-100 based buffers (0.5-1%) maintain most protein-protein interactions

    • RIPA buffer may disrupt some interactions but reduces background

    • For weaker interactions, use gentler detergents like digitonin (0.5-1%)

  • Salt concentration adjustments:

    • Standard: 150mM NaCl

    • Lower salt (50-100mM) preserves weaker interactions

    • Higher salt (250-500mM) reduces non-specific binding

• Cross-linking considerations:

  • For transient interactions, consider mild cross-linking with DSP or formaldehyde

  • Cross-linking preserves complexes but may reduce antibody accessibility to epitopes

  • Requires careful optimization of cross-linker concentration and reaction time

• Pre-clearing strategy:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Include isotype-matched control antibodies to identify false positives

  • For rabbit polyclonal antibodies like 29902-1-AP, use normal rabbit IgG as control

• Antibody immobilization approaches:

  • Direct method: Add antibody to lysate, then capture with protein A/G beads

  • Pre-coupling method: Immobilize antibody on beads before adding to lysate

  • Pre-coupling often reduces co-IP of antibody heavy/light chains in downstream analysis

• Washing optimization:

  • Balance stringency (reducing background) with complex preservation

  • Consider sequential washes with decreasing stringency

  • For PFKFB4-FBXO28 interaction , moderate stringency washing conditions are appropriate

• Elution methods:

  • Denaturing elution (SDS, heat): Disrupts all interactions, maximizes yield

  • Native elution (excess peptide): Preserves complex integrity for functional studies

  • Choose based on downstream applications

• Validation strategies:

  • Reverse IP: Use FBXO28 antibody to pull down, then detect PFKFB4

  • Include positive controls (known interaction partners)

  • Mass spectrometry analysis to identify novel interaction partners

  • This approach successfully identified FBXO28 as a PFKFB4 interaction partner

• Analyzing ubiquitination:

  • For studying PFKFB4's role in HIF-1α ubiquitination :

    • Use denaturing conditions (1% SDS, boiling) to disrupt non-covalent interactions

    • IP HIF-1α, then probe for ubiquitin

    • Compare ubiquitination patterns with and without PFKFB4 manipulation

By optimizing these parameters, researchers can effectively use PFKFB4 antibodies to isolate protein complexes and investigate novel interaction networks, potentially revealing additional non-canonical functions beyond metabolic regulation.

What are the methodological considerations for using PFKFB4 antibodies in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence using PFKFB4 antibodies enables simultaneous visualization of multiple markers within the same sample, providing valuable spatial context for PFKFB4 expression and co-localization patterns. Researchers should address these methodological considerations:

• Antibody compatibility assessment:

  • Verify PFKFB4 antibody host species (typically rabbit for available antibodies)

  • Select companion antibodies from different host species to avoid cross-reactivity

  • For same-species antibodies, consider directly conjugated primary antibodies or sequential staining protocols

• Optimal fixation and antigen retrieval:

  • For PFKFB4 detection, both formalin-fixed paraffin-embedded (FFPE) and frozen sections are compatible

  • For FFPE sections with PFKFB4 antibodies:

    • Primary recommendation: TE buffer at pH 9.0

    • Alternative: citrate buffer at pH 6.0

  • Optimize retrieval conditions to preserve both PFKFB4 and co-targeted antigens

• Signal amplification strategies:

  • For low-abundance targets, consider tyramide signal amplification (TSA)

  • TSA allows use of primary antibodies from the same species without cross-reactivity

  • Enables sequential multiplexing with PFKFB4 antibodies

• Multi-panel design for cancer metabolism studies:

  • PFKFB4 + cell type markers (e.g., cytokeratins, CD45, CD31)

  • PFKFB4 + other metabolic enzymes (e.g., PKM2, LDHA, G6PD)

  • PFKFB4 + hypoxia markers (HIF-1α, CA9) to investigate the relationship identified in previous research

  • PFKFB4 + FBXO28 to examine co-localization of these interaction partners

• Signal separation considerations:

  • Select fluorophores with minimal spectral overlap

  • Include single-stained controls for spectral unmixing

  • Consider automated multispectral imaging platforms for complex panels

• Quantification approaches:

  • Cell-by-cell quantification of PFKFB4 intensity

  • Co-localization analysis with interaction partners like FBXO28

  • Spatial relationship to microenvironmental features (hypoxic regions, vasculature)

• Validation controls:

  • Include tissues known to be positive for PFKFB4:

    • Human kidney tissue

    • Mouse testis tissue

    • Cancer cell lines (HUVEC cells are positive for IF/ICC)

  • Include negative controls (primary antibody omission, isotype controls)

  • Consider PFKFB4 knockdown controls in cell line studies

• Image analysis and quantification:

  • Use appropriate software for multi-parameter analysis (HALO, QuPath, etc.)

  • Establish consistent thresholds for positive staining

  • For spatial analysis, consider nearest neighbor calculations or distance mapping

Successful implementation of these methodological considerations will enable researchers to contextualize PFKFB4 expression within the complex tumor microenvironment, potentially revealing cell type-specific expression patterns and functional relationships with metabolic pathways and hypoxia signaling networks.

How does PFKFB4 antibody-based research contribute to our understanding of cancer metabolism and potential therapeutic strategies?

PFKFB4 antibody-based research has significantly advanced our understanding of cancer metabolism and identified promising therapeutic opportunities through several key contributions:

• Revealing cancer-specific metabolic adaptations:

  • Immunohistochemical studies using PFKFB4 antibodies have demonstrated significantly elevated expression in multiple cancer types compared to normal tissues

  • This differential expression pattern has been documented in:

    • Prostate cancer versus benign prostatic hyperplasia (p < 0.000)

    • Prostate cancer versus adjacent normal tissue (p < 0.001)

    • Lung cancer compared to matched normal control tissue

  • These findings establish PFKFB4 as a potential cancer-specific metabolic vulnerability

• Uncovering dual functions in metabolism and signaling:

  • Antibody-based techniques have revealed PFKFB4's role beyond glycolytic regulation

  • Immunoprecipitation followed by mass spectrometry identified FBXO28 as a novel PFKFB4 interaction partner

  • This interaction mediates HIF-1α ubiquitylation and degradation, connecting metabolic regulation with hypoxia signaling

  • The discovery highlights how metabolic enzymes can directly influence oncogenic signaling pathways

• Tracking therapy resistance mechanisms:

  • PFKFB4 antibody staining has demonstrated increased expression during the development of castration resistance in prostate cancer

  • This suggests PFKFB4-mediated metabolic reprogramming contributes to therapy resistance

  • The finding provides rationale for combining PFKFB4 inhibitors with existing therapies to prevent resistance

• Enabling biomarker development:

  • PFKFB4 has been identified as a promising biomarker for poor prognosis in gastric cancer

  • Standardized immunohistochemical protocols using validated PFKFB4 antibodies could potentially translate to clinical biomarker applications

  • This may help stratify patients for more aggressive treatment or targeted therapy approaches

• Identifying therapeutic opportunities:

  • The cancer-specific expression pattern of PFKFB4 makes it an attractive therapeutic target

  • Its newly discovered role in HIF-1α regulation provides an additional mechanism through which PFKFB4 inhibition might exert anti-cancer effects

  • PFKFB4 antibodies enable target validation in preclinical models and potential companion diagnostic development