PFKP Antibody

What is PFKP and why is it important in metabolic research?

PFKP is the platelet isoform of phosphofructokinase, a key regulatory enzyme in glycolysis. In humans, the canonical protein consists of 784 amino acid residues with a mass of 85.6 kDa, primarily localized in the cytoplasm . As a member of the Phosphofructokinase type A (PFKA) protein family, PFKP is critical for energy metabolism research because it catalyzes the rate-limiting step of glycolysis. It's particularly important in cancer research due to the Warburg effect, where cancer cells show increased glycolysis even in the presence of oxygen.

The protein has several synonyms including PFK-C, PFKF, ATP-PFK, and 6-phosphofructokinase type C . Orthologs have been identified in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken, making it relevant for comparative metabolic studies across species .

Which applications are most commonly used for PFKP antibody detection?

PFKP antibodies are versatile tools employed across multiple experimental applications:

When selecting a PFKP antibody, researchers should verify that it has been validated for their specific application of interest, as performance can vary significantly between applications .

How do I select the appropriate PFKP antibody for my research?

Selection of the appropriate PFKP antibody depends on several factors:

• Species reactivity: Confirm that the antibody reacts with your experimental species. Many PFKP antibodies react with human, mouse, and rat samples, but cross-reactivity varies between products .

• Application compatibility: Verify the antibody has been validated for your specific application. For example, antibody ab119796 is suitable for WB, IHC-P, ICC/IF, and Flow Cytometry .

• Clonality considerations:

  • Monoclonal antibodies (e.g., OTI1D6, OTI1F2) offer high specificity and reproducibility .

  • Polyclonal antibodies may provide better sensitivity for low-abundance targets but potentially lower specificity .

• Immunogen location: Consider which region of PFKP the antibody targets. For example:

  • Some antibodies target the N-terminal region (aa 1-50)

  • Others target the C-terminal region (aa 605-784)

  • Some target middle regions (aa 350 to C-terminus)

• Validation data: Review published validation data, including Western blot images showing the expected 85-86 kDa band and citations in peer-reviewed literature .

What are the optimal conditions for Western blotting with PFKP antibodies?

For optimal Western blotting results with PFKP antibodies:

Sample Preparation:

• Use cell lines with known PFKP expression (positive controls) such as HeLa, 293T, Jurkat, U-87 MG, or BxPC-3 cells .

• Prepare whole cell lysates with complete protease inhibitors to prevent degradation.

• Load 20-50 μg of total protein per lane .

Antibody Conditions:

• Primary antibody dilutions typically range from 1:500 to 1:2000 depending on the specific antibody .

• For Abcam ab186132: Use 0.1 μg/mL concentration .

• For Cell Signaling Technology #5412: Use 1:1000 dilution .

Detection Parameters:

• Expected molecular weight: 85-86 kDa (some migration variation may occur between gel systems) .

• ECL detection systems provide adequate sensitivity for endogenous PFKP detection .

• Exposure times vary by sample but typically range from 1-3 minutes .

Troubleshooting Tips:

• If detecting multiple bands, optimize antibody concentration and blocking conditions.

• If signal is weak, increase antibody concentration or extend incubation time.

• PFKP knockout cell lysates (e.g., ab257580) can serve as valuable negative controls to confirm specificity .

How can I optimize immunohistochemistry protocols for PFKP detection in cancer tissues?

Optimizing IHC protocols for PFKP detection in cancer tissues requires careful attention to several parameters:

Tissue Processing:

• Formalin-fixed paraffin-embedded (FFPE) tissues work well for most PFKP antibodies.

• Freshly prepared sections (4-6 μm thick) yield optimal results.

• For some antibodies (e.g., ab119796), antigen retrieval is critical .

Antigen Retrieval Methods:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is commonly effective.

• Optimal retrieval conditions may vary by antibody and should be empirically determined.

Antibody Dilution and Incubation:

• Start with manufacturer's recommended dilution (typically 1:50 to 1:200).

• Incubate at 4°C overnight or at room temperature for 1-2 hours.

• For ab119796, which has been cited in 15 publications, follow specific manufacturer protocols .

Signal Detection:

• Both chromogenic (DAB) and fluorescent detection methods work well.

• For multiplex staining (e.g., with other glycolytic enzymes), fluorescent detection offers advantages.

• When interpreting results, note that PFKP shows primarily cytoplasmic localization .

Controls:

• Include positive controls from tissues known to express PFKP (e.g., certain cancer types).

• Use PFKP-knockout tissues or cells as negative controls when available.

• Consider including normal adjacent tissue for comparison in cancer studies.

What considerations are important when using PFKP antibodies for cancer research?

When utilizing PFKP antibodies in cancer research, several important considerations should be addressed:

Expression Patterns:

• PFKP is frequently upregulated in various cancers, including head and neck squamous cell carcinoma (HNSCC) .

• Expression levels can vary significantly between cancer types and even within tumor regions.

• PFKP expression correlates with poor prognosis in several cancer types, making it a potential biomarker .

Regulatory Mechanisms:

• A positive feedback loop exists between PFKP and c-Myc in HNSCC, driving cancer progression .

• This relationship involves ERK-mediated stability of c-Myc and c-Myc-stimulated PFKP expression at the transcriptional level.

Experimental Design:

• Include appropriate cancer cell lines as controls (e.g., HeLa, U-87 MG, BxPC-3, Jurkat) .

• Consider dual staining with other metabolic markers to assess glycolytic phenotypes.

• For studying PFKP-c-Myc interactions, dual-luciferase reporter assays can be employed as described by researchers studying HNSCC .

Therapeutic Implications:

• Targeting PFKP alone or in combination with other metabolic enzymes may represent a therapeutic strategy.

• Research indicates that co-targeting PFKP and c-Myc triggers synergistic anti-tumor effects in HNSCC .

• When designing such studies, antibody specificity becomes particularly critical to distinguish PFKP from other PFK isoforms.

How can I use PFKP antibodies to investigate cancer metabolism and the Warburg effect?

PFKP antibodies can be powerful tools for investigating the Warburg effect and cancer metabolism:

Metabolic Profiling:

• Use PFKP antibodies in conjunction with other glycolytic enzyme antibodies (HK2, PKM2, LDHA) to characterize the glycolytic profile of cancer cells.

• Western blotting and IHC can establish baseline expression levels across different cancer types or stages.

Metabolic Adaptation Studies:

• Monitor PFKP expression changes under varying oxygen conditions (normoxia vs. hypoxia) to study metabolic adaptation.

• Combine with functional glycolysis assays (e.g., Seahorse analyzer) to correlate PFKP expression with glycolytic flux.

Methodological Approach:

• Establish baseline PFKP expression in your cancer model using validated antibodies

• Manipulate conditions (hypoxia, glucose availability, oncogene activation)

• Assess PFKP expression changes via Western blot (1:1000 dilution recommended for most antibodies)

• Correlate with functional metabolic parameters

• Consider subcellular localization via immunofluorescence as PFKP function may be affected by localization

Tumor Heterogeneity Analysis:

• Use immunofluorescence with PFKP antibodies to map metabolic heterogeneity within tumors.

• Multiple antibodies are validated for IF applications, including products from Abcam and Boster Bio .

• Combine with markers of hypoxia (HIF-1α) or proliferation (Ki-67) for contextual analysis.

What are the considerations for using PFKP antibodies in immunological and drug resistance studies?

PFKP antibodies can provide valuable insights into immunological processes and drug resistance mechanisms:

Immune Cell Metabolism:

• Different immune cell populations exhibit varying metabolic profiles during activation and effector functions.

• PFKP antibodies can help characterize glycolytic reprogramming in immune cells during inflammation or cancer.

• Flow cytometry applications (available with antibodies like ab119796) allow for cell-specific metabolic profiling .

Drug Resistance Assessment:

• Research indicates PFKP may contribute to drug resistance in cancer .

• Monitor PFKP expression changes before and after drug treatment using Western blot.

• For correlation analysis between PFKP expression and drug sensitivity:

  • Extract protein from drug-sensitive and resistant cell populations

  • Perform Western blot using optimized PFKP antibody dilutions (typically 1:1000)

  • Quantify relative expression levels

  • Correlate with IC50 values from drug sensitivity assays

Methodological Considerations:

• When studying immune cell populations, consider using flow cytometry-validated PFKP antibodies.

• For drug resistance studies, consistent sample preparation is crucial for reliable quantitative comparisons.

• The oncoPredict package can be used to predict drug responses based on PFKP expression levels .

Combination Studies:

• Co-staining with markers of drug resistance (e.g., MDR1, ABCG2) can provide mechanistic insights.

• Immunoprecipitation with PFKP antibodies (using antibodies validated for IP, such as Cell Signaling #5412) can identify novel interacting partners in drug-resistant contexts .

How do I use PFKP antibodies for prognostic and diagnostic biomarker research?

PFKP shows promise as both a prognostic and diagnostic biomarker across multiple cancer types:

Biomarker Validation Strategy:

• Tissue Microarray Analysis:

  • Use IHC-validated PFKP antibodies (such as ab119796 or A07337-2) on tissue microarrays .

  • Score expression patterns (0-3+) based on staining intensity and percentage of positive cells.

  • Correlate with patient outcome data for prognostic value assessment.

• Receiver Operating Characteristic (ROC) Analysis:

  • Recent research shows PFKP has diagnostic value across multiple cancers .

  • ROC curve analysis methodology:

    • Calculate sensitivity and specificity at various PFKP expression thresholds

    • Generate AUC values (AUC 0.75-1.0 indicates high diagnostic value)

    • Implement using R/Bioconductor package pROC as described in published protocols

Correlation Analysis:

• PFKP expression correlates with tumor mutational burden (TMB) in some cancers.

• Methodology for TMB correlation:

  • Extract TMB data using packages like TCGAbiolinks

  • Perform correlation analysis using ggstatsplot and ggplot2 packages

  • Generate visualization of PFKP expression vs. TMB

Multiparameter Analysis:

• Combine PFKP with other metabolic markers for improved prognostic value.

• Consider immune infiltration metrics alongside PFKP expression:

  • Immune cell infiltration can be assessed using CIBERSORT algorithm

  • Pearson correlation coefficients between PFKP and 18 immune cell types provide insights into tumor immunology

How can I resolve specificity issues when working with PFKP antibodies?

Specificity challenges with PFKP antibodies require systematic troubleshooting:

Common Specificity Issues:

• Cross-reactivity with other PFK isoforms (PFKL, PFKM)

• Non-specific binding resulting in multiple bands in Western blot

• Background staining in IHC/ICC

Validation Approaches:

• Knockout/knockdown controls:

  • Use PFKP knockout cell lysates (e.g., ab257580) as negative controls .

  • Compare with wild-type samples to identify specific bands/staining.

  • If knockout cells are unavailable, siRNA-mediated knockdown provides an alternative.

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (when available from manufacturers).

  • Specific signals should be blocked by peptide competition.

  • Some suppliers offer blocking peptides that can be purchased separately .

• Multiple antibody validation:

  • Test multiple antibodies targeting different epitopes of PFKP.

  • Consistent results across antibodies increase confidence in specificity.

  • Consider combinations like ab186132 (N-terminal epitope) and ab137636 (C-terminal) .

Optimizing Experimental Conditions:

• Increase blocking stringency (5% BSA or milk, longer blocking times).

• Optimize antibody dilutions (test range from 1:500 to 1:2000).

• For Western blots, more stringent washing conditions can reduce non-specific binding.

• For IHC/ICC, include additional blocking steps (e.g., avidin/biotin blocking for biotin-based detection systems).

What are the best practices for quantitative analysis of PFKP expression in experimental samples?

Quantitative analysis of PFKP expression requires rigorous methodological approaches:

Western Blot Quantification:

• Sample Preparation Consistency:

  • Standardize lysis buffers and extraction protocols.

  • Determine protein concentration using reliable methods (BCA or Bradford).

  • Load equal amounts of protein (typically 20-50 μg) per lane .

• Internal Controls:

  • Always include loading controls (β-actin, GAPDH, total protein).

  • Consider the use of recombinant PFKP protein standards for absolute quantification.

  • Include reference cell lines with known PFKP expression levels (e.g., HeLa, Jurkat) .

• Image Acquisition and Analysis:

  • Capture images within the linear dynamic range of your detection system.

  • Use software like ImageJ or specialized Western blot analysis software.

  • Normalize PFKP band intensity to loading controls.

  • Report relative fold changes compared to appropriate baseline conditions.

IHC/IF Quantification:

• Standardized Scoring Systems:

  • Implement H-score methodology (staining intensity × percentage of positive cells).

  • Consider automated image analysis for reproducibility.

  • Use digital pathology approaches when available.

• Controls for Staining Variability:

  • Include control tissues on each slide to normalize between batches.

  • Use automated staining platforms when possible to reduce technical variability.

  • Blind observers to experimental conditions during scoring.

Flow Cytometry Quantification:

• Standardization Methods:

  • Use antibody capture beads for setup and calibration.

  • Include fluorescence minus one (FMO) controls.

  • Consider using quantification beads to convert MFI to absolute antibody binding capacity.

• Analysis Approaches:

  • Report median fluorescence intensity (MFI) rather than mean.

  • Calculate fold change relative to appropriate controls.

  • Consider using histogram overlay analysis for population shifts.

How can PFKP antibodies be effectively used in multi-parameter studies and high-throughput analyses?

Integrating PFKP antibodies into multi-parameter and high-throughput studies requires specialized approaches:

Multiplexed Immunofluorescence:

• Antibody Panel Design:

  • Combine PFKP antibodies with other metabolic markers (e.g., HK2, LDHA).

  • Test for antibody compatibility (species, isotype, fluorophore spectral overlap).

  • For cancer research, consider combining with proliferation markers (Ki-67) or hypoxia markers (HIF-1α).

• Sequential Staining Protocols:

  • For same-species antibodies, consider tyramide signal amplification methods.

  • Test order of antibody application to optimize signal-to-noise ratio.

  • Include appropriate controls for each marker in the panel.

High-Content Screening:

• Assay Development:

  • Optimize cell density, fixation conditions, and antibody concentrations.

  • Validate using positive and negative controls (e.g., PFKP modulation by siRNA).

  • Establish robust quantification parameters (intensity, localization, morphology).

• Data Analysis Approaches:

  • Implement machine learning algorithms for phenotypic classification.

  • Consider multivariate analysis methods to integrate PFKP data with other parameters.

  • Validation strategies should include orthogonal techniques (e.g., Western blot).

Reverse Phase Protein Array (RPPA):

• Antibody Validation for RPPA:

  • Verify antibody specificity and sensitivity in Western blot before RPPA.

  • Test linear dynamic range using dilution series.

  • Include positive and negative controls on each array.

• Data Normalization Strategies:

  • Apply total protein normalization methods.

  • Consider slide-to-slide normalization using reference samples.

  • Integrate with public RPPA datasets for comparative analysis.

Integration with -Omics Data:

• Correlate PFKP protein levels with transcriptomic data.

• Integrate with metabolomic profiles to assess glycolytic flux.

• Use pathway analysis tools to contextualize PFKP within broader metabolic networks.

• Recent research has demonstrated the value of integrating PFKP expression data with immune cell infiltration metrics and drug sensitivity predictions .

What are the latest applications of PFKP antibodies in cancer immunotherapy research?

PFKP antibodies are increasingly used in cancer immunotherapy research due to emerging connections between metabolism and immune function:

Tumor-Immune Microenvironment Analysis:

• Recent studies show PFKP expression correlates with immune cell infiltration across multiple cancer types .

• PFKP antibodies can be used in multiplex immunofluorescence to simultaneously visualize metabolic states and immune cell populations.

• Methodology involves co-staining tissue sections with PFKP antibodies and immune cell markers (CD8, CD4, CD68).

Metabolic Competition Assessment:

• Cancer cells and T cells compete for glucose in the tumor microenvironment.

• PFKP antibodies help quantify glycolytic capacity in both tumor and infiltrating immune cells.

• Flow cytometry with PFKP antibodies can assess relative expression between cell populations.

Checkpoint Inhibitor Response Prediction:

• Correlation between PFKP expression and PD-L1 or tumor mutational burden (TMB) may predict immunotherapy response .

• Recent research methodologies include:

  • ROC curve analysis of PFKP across cancers using R/Bioconductor package pROC

  • Correlation analysis with TMB data obtained via TCGAbiolinks

  • Integration with immune cell infiltration data from CIBERSORT algorithm

Therapeutic Target Assessment:

• The PFKP/c-Myc positive feedback loop represents a potential therapeutic target .

• Dual targeting of PFKP and c-Myc shows synergistic anti-tumor effects in HNSCC .

• Antibodies are essential tools for validating target engagement in preclinical models.

How can I use PFKP antibodies to study the relationship between metabolism and cellular differentiation?

PFKP antibodies offer valuable insights into metabolic changes during cellular differentiation:

Stem Cell Differentiation:

• Glycolytic metabolism often predominates in stem cells, with shifts occurring during differentiation.

• PFKP antibodies can track metabolic reprogramming during differentiation processes.

• Methodology:

  • Collect cells at different differentiation stages

  • Perform Western blot using optimized PFKP antibody dilutions (typically 1:1000)

  • Correlate PFKP expression with differentiation markers

  • Complement with functional glycolysis assays

Cancer Stem Cell Analysis:

• Cancer stem cells often exhibit distinct metabolic profiles.

• PFKP expression in cancer stem cell populations can be assessed using:

  • Flow cytometry with PFKP antibodies on sorted stem cell populations

  • Immunofluorescence co-staining with stem cell markers (CD44, CD133)

  • Western blot analysis of stem cell-enriched vs. differentiated populations

Lineage-Specific Metabolic Patterns:

• Different cell lineages exhibit varying dependence on glycolysis.

• Immunohistochemistry with PFKP antibodies can map metabolic heterogeneity during development.

• Compare PFKP expression across differentiation stages using antibodies validated for multiple species to enable developmental studies .

Experimental Design Considerations:

• Include appropriate controls for each differentiation stage.

• Consider using multiple antibodies targeting different epitopes of PFKP for validation.

• Correlate protein-level changes (via antibody detection) with enzymatic activity assays.

• Integrate with transcriptomic and epigenetic data for comprehensive mechanistic insights.

What considerations are important when using PFKP antibodies in metastasis and invasion studies?

PFKP has emerging roles in cancer metastasis and invasion, making antibodies against this protein valuable for such studies:

Metastasis Model Systems:

• Recent studies show elevated PFKP facilitates angiogenesis and metastasis in HNSCC .

• PFKP antibodies can be used to:

  • Compare expression between primary tumors and metastatic lesions

  • Assess expression in circulating tumor cells

  • Evaluate changes during epithelial-mesenchymal transition (EMT)

Migration and Invasion Assays:

• For cells with manipulated PFKP levels, antibodies confirm successful modulation.

• Immunofluorescence can assess subcellular localization during migration.

• Methodology:

  • Perform transwell migration/invasion assays

  • Fix and stain cells with PFKP antibodies

  • Correlate PFKP expression/localization with migratory capacity

  • Consider co-staining with cytoskeletal markers (F-actin, tubulin)

Metabolic Adaptation During Metastasis:

• Metastasizing cells must adapt their metabolism to survive in new environments.

• PFKP antibodies can track metabolic changes during the metastatic cascade.

• Consider combining with hypoxia markers, as metastasizing cells often encounter hypoxic conditions.

In Vivo Metastasis Studies:

• IHC-validated antibodies can assess PFKP expression in xenograft models.

• Serial sections allow correlation with other markers (proliferation, angiogenesis).

• For mouse models, ensure the selected antibody has validated cross-reactivity with mouse PFKP .

Mechanistic Studies:

• The PFKP/c-Myc feedback loop drives metastasis in some cancers .

• Immunoprecipitation with PFKP antibodies can identify novel interaction partners.

• For such studies, select antibodies specifically validated for IP applications, such as Cell Signaling #5412 .