PFP-BETA1 Antibody

What is PFP-BETA1 and what role does it play in plant metabolism?

PFP-BETA1 is the catalytic subunit of pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP), an enzyme that catalyzes the phosphorylation of D-fructose 6-phosphate, representing the first committing step of glycolysis in plants. Unlike conventional ATP-dependent phosphofructokinases, PFP uses inorganic pyrophosphate (PPi) as a phosphoryl donor instead of ATP, which renders the reaction reversible. This biochemical characteristic enables PFP to function bidirectionally in both glycolysis and gluconeogenesis pathways, providing plants with metabolic flexibility, particularly under energy-limited conditions .

The reversibility of this reaction is particularly significant in plant physiology as it allows for adaptation to changing environmental conditions and energy states. The enzyme's ability to operate in both anabolic and catabolic directions makes it a crucial regulatory point in carbohydrate metabolism, especially when plants face stress conditions that may limit ATP availability.

How does the specificity of PFP-BETA1 antibody compare to other metabolic enzyme antibodies?

PFP-BETA1 antibody is a rabbit polyclonal antibody raised against a specific recombinant Arabidopsis thaliana PFP-BETA1 protein fragment (amino acids 157-427) . Its specificity differs from other metabolic enzyme antibodies in several key aspects:

• Target recognition: While antibodies against ATP-dependent phosphofructokinases typically recognize conserved ATP-binding domains, PFP-BETA1 antibody targets regions specific to PPi-utilizing enzymes.

• Cross-reactivity profile: The antibody has been optimized for plant samples, whereas many metabolic antibodies are primarily designed for mammalian systems.

• Epitope characteristics: The immunogen used (amino acids 157-427) likely encompasses catalytic regions specific to the PPi-dependent mechanism, making this antibody suitable for studying this alternative glycolytic pathway.

When designing experiments, researchers should be mindful that the specificity of this antibody makes it particularly valuable for comparative studies between conventional ATP-dependent glycolysis and PPi-dependent pathways in plants.

What are the validated applications for PFP-BETA1 antibody in plant research?

The PFP-BETA1 polyclonal antibody has been validated for several experimental applications in plant research, with ELISA and Western Blot being the primary validated methodologies . For each application, specific protocols have been optimized:

Western Blot Protocol:

• Sample preparation: Extract plant proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitors.

• Protein separation: Load 20-50 μg of protein per lane on SDS-PAGE.

• Transfer: Use standard transfer conditions (100V for 1 hour or 30V overnight).

• Blocking: Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute PFP-BETA1 antibody 1:1000 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG (1:5000) for 1 hour at room temperature.

• Detection: Visualize using ECL substrate with expected band size corresponding to PFP-BETA1.

ELISA Protocol:

• Coating: Coat wells with target antigen (plant extract or purified protein) overnight at 4°C.

• Blocking: Block with 1% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Apply diluted PFP-BETA1 antibody (1:2000) for 2 hours at room temperature.

• Secondary antibody: Use HRP-conjugated detection antibody for 1 hour.

• Development: Add TMB substrate and measure absorbance at 450 nm.

These validated methods provide researchers with a solid foundation for investigating PFP-BETA1 expression and function in plant systems.

How should samples be prepared to optimize PFP-BETA1 detection in plant tissues?

Optimal sample preparation is critical for successfully detecting PFP-BETA1 in plant tissues. The following methodological approach is recommended based on enzyme characteristics and antibody properties:

• Tissue selection: Choose metabolically active tissues where glycolysis is prominent (e.g., developing seeds, germinating seedlings, or roots).

• Extraction buffer composition:

  • 50 mM HEPES-KOH (pH 7.5)

  • 10 mM MgCl₂ (to maintain enzyme structure)

  • 1 mM EDTA (to chelate heavy metals)

  • 5 mM DTT (to maintain reducing environment)

  • 10% glycerol (for stability)

  • 1% PVPP (to remove phenolic compounds)

  • Protease inhibitor cocktail (to prevent degradation)

• Extraction procedure:

  • Grind tissue in liquid nitrogen to a fine powder

  • Add 4 volumes of cold extraction buffer

  • Homogenize thoroughly

  • Centrifuge at 15,000 × g for 15 minutes at 4°C

  • Collect supernatant for analysis

• Sample storage: Aliquot samples and store at -80°C with 10% glycerol to prevent freeze-thaw cycles that may denature the protein .

This preparation method preserves both the native conformation of PFP-BETA1 and its enzymatic activity, which can be valuable for correlating immunological detection with functional assays.

What are common issues when using PFP-BETA1 antibody in Western blotting and how can they be addressed?

Researchers commonly encounter several challenges when using PFP-BETA1 antibody in Western blotting experiments. The following methodological approaches can help address these issues:

When optimizing Western blot protocols for PFP-BETA1 detection, it's important to remember that this enzyme functions in a complex with other subunits. Therefore, sample preparation methods that preserve protein-protein interactions might be beneficial for certain research questions, while denaturing conditions may be preferable for others depending on the specific epitope recognized by the antibody .

How can researchers validate the specificity of PFP-BETA1 antibody in their experimental systems?

Validating antibody specificity is critical for ensuring reliable and reproducible results when studying PFP-BETA1. Researchers should implement the following multi-step validation protocol:

• Positive and negative controls:

  • Positive control: Use Arabidopsis thaliana extracts where PFP-BETA1 is well-characterized

  • Negative control: Include samples from tissues with minimal PFP-BETA1 expression or knockout/knockdown lines if available

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (Arabidopsis thaliana PFP-BETA1 protein fragment, amino acids 157-427)

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific signals should disappear in the peptide-blocked samples

• Molecular weight verification:

  • Confirm that the detected band matches the expected molecular weight

  • PFP-BETA1 should appear at its predicted size based on amino acid sequence

• Correlation with functional data:

  • Compare antibody detection levels with enzyme activity measurements

  • Tissues with higher PFP activity should show stronger immunoreactivity

• Orthogonal detection methods:

  • Verify protein expression using alternative techniques such as mass spectrometry

  • Correlate with mRNA expression using RT-PCR or RNA-seq data

By implementing this comprehensive validation approach, researchers can confidently attribute their observations to genuine PFP-BETA1 detection rather than non-specific interactions.

How can PFP-BETA1 antibody be used to investigate metabolic reprogramming during plant stress responses?

PFP-BETA1 antibody serves as a powerful tool for investigating metabolic reprogramming during various plant stress responses. The following methodological approach can be implemented:

• Experimental design for stress studies:

  • Subject plants to relevant stresses (drought, salt, cold, hypoxia)

  • Collect samples at multiple time points (0, 6, 12, 24, 48 hours)

  • Process parallel samples for protein analysis and metabolite profiling

• Multi-level analysis methodology:

  • Western blot with PFP-BETA1 antibody to quantify protein expression changes

  • Enzyme activity assays to correlate expression with functional changes

  • Immunolocalization to detect subcellular redistribution during stress

  • Co-immunoprecipitation to identify stress-induced protein interaction partners

• Correlation with metabolic indicators:

  • Measure glycolytic intermediates (especially fructose-6-phosphate and fructose-1,6-bisphosphate)

  • Assess PPi and ATP levels to understand energy status

  • Monitor end products to evaluate pathway flux changes

This approach allows researchers to determine how plants modulate the balance between ATP-dependent and PPi-dependent glycolysis during energy-limited conditions, potentially revealing adaptive mechanisms where PFP-BETA1 activity becomes particularly important due to its lower energy requirements compared to ATP-dependent phosphofructokinases .

What experimental approaches can combine immunoprecipitation with PFP-BETA1 antibody and proteomics to identify novel interaction partners?

Identifying novel interaction partners of PFP-BETA1 can provide valuable insights into the regulation and integration of plant glycolysis with other cellular processes. The following methodological workflow combines immunoprecipitation with advanced proteomics:

• Sample preparation for immunoprecipitation:

  • Prepare native protein extracts using gentle lysis buffers (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors)

  • Clear lysates by centrifugation (20,000 × g, 15 min, 4°C)

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

• Immunoprecipitation protocol:

  • Incubate cleared lysates with PFP-BETA1 antibody (5 μg) overnight at 4°C with gentle rotation

  • Add protein G magnetic beads and incubate for 2-3 hours

  • Wash extensively with decreasing salt concentrations

  • Perform parallel control IPs with pre-immune serum or IgG

• Mass spectrometry analysis:

  • Elute proteins directly in MS-compatible buffer

  • Perform on-bead tryptic digestion

  • Analyze peptides using LC-MS/MS

  • Process data using appropriate search algorithms

• Validation of novel interactions:

  • Perform reciprocal co-IPs with antibodies against identified partners

  • Use proximity ligation assays for in situ confirmation

  • Verify functional relevance through enzyme activity assays

• Interaction network construction:

  • Map identified proteins to metabolic pathways

  • Create interaction networks using bioinformatics tools

  • Compare networks across different physiological conditions

This integrated approach allows researchers to move beyond the conventional understanding of PFP-BETA1 as merely a glycolytic enzyme and potentially discover new regulatory mechanisms and metabolic integration points .

How can researchers utilize PFP-BETA1 antibody to investigate the evolutionary conservation of PPi-dependent glycolysis across plant species?

Investigating the evolutionary conservation of PPi-dependent glycolysis across plant species requires a carefully designed comparative approach using PFP-BETA1 antibody. The following methodological framework is recommended:

• Species selection strategy:

  • Include representatives from major plant lineages (bryophytes, lycophytes, gymnosperms, angiosperms)

  • Select species from diverse ecological niches (aquatic, mesic, xeric)

  • Include both C3 and C4 photosynthetic plants

  • Consider species with sequenced genomes for correlative analyses

• Cross-reactivity assessment:

  • Perform initial Western blots on all species using identical protein amounts

  • Document band patterns, intensities, and molecular weights

  • Optimize antibody concentration for cross-reactive detection

• Comparative immunological analysis:

  • Standardize protein loading using conserved housekeeping proteins

  • Quantify relative PFP-BETA1 abundance across species

  • Correlate expression levels with phylogenetic relationships

• Structure-function correlation:

  • Combine immunodetection with enzyme activity measurements

  • Calculate specific activity (activity per unit of immunodetectable protein)

  • Correlate variations in activity with environmental adaptations

• Epitope conservation analysis:

  • Perform in silico analysis of epitope conservation based on sequence alignments

  • Identify conserved vs. variable regions within the antibody binding site

  • Use this information to interpret cross-reactivity patterns

This comprehensive approach enables researchers to trace the evolutionary history of PPi-dependent glycolysis and potentially identify adaptive modifications in different plant lineages, providing insights into the selective pressures that have shaped central carbon metabolism throughout plant evolution .

How can PFP-BETA1 antibody be combined with microscopy techniques to study subcellular localization?

Combining PFP-BETA1 antibody with advanced microscopy techniques provides valuable insights into the subcellular localization and potential compartmentalization of glycolytic enzymes in plant cells. The following methodological approach is recommended:

• Sample preparation for immunolocalization:

  • Fix plant tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours

  • Embed in appropriate medium (paraffin for light microscopy, resin for electron microscopy)

  • Section at optimal thickness (5-10 μm for light microscopy, 70-100 nm for electron microscopy)

  • Perform antigen retrieval if necessary (citrate buffer, pH 6.0, 95°C for 10 minutes)

• Immunofluorescence protocol:

  • Block sections with 3% BSA in PBS for 1 hour

  • Incubate with PFP-BETA1 antibody (1:100-1:500 dilution) overnight at 4°C

  • Apply fluorophore-conjugated secondary antibody (anti-rabbit IgG)

  • Counterstain with organelle-specific markers:

    • Chloroplasts: Autofluorescence

    • Mitochondria: MitoTracker dyes

    • Cytoskeleton: Phalloidin for F-actin

    • Nuclei: DAPI

  • Image using confocal laser scanning microscopy

• Immuno-electron microscopy:

  • Incubate ultrathin sections with PFP-BETA1 antibody

  • Apply gold-conjugated secondary antibody

  • Quantify gold particle distribution across cellular compartments

• Advanced imaging approaches:

  • Super-resolution microscopy (STED, PALM, or STORM) for precise localization

  • FRET microscopy if combining with other fluorescently-labeled molecules

  • Live-cell imaging using microinjection of fluorescently-labeled antibodies

This integrated microscopy approach can reveal whether PFP-BETA1 is strictly cytosolic or associates with particular organelles, potentially uncovering previously unrecognized metabolic compartmentalization that may be physiologically significant for the regulation of plant glycolysis .

What experimental design would allow researchers to correlate PFP-BETA1 expression with enzyme activity in different plant tissues?

A comprehensive experimental design correlating PFP-BETA1 expression with enzyme activity requires integrated biochemical and immunological approaches. The following methodology enables researchers to establish meaningful structure-function relationships:

• Tissue sampling strategy:

  • Collect diverse plant tissues (roots, stems, leaves, flowers, seeds)

  • Sample at different developmental stages

  • Include tissues under various environmental conditions

  • Process all samples in parallel for both protein detection and enzyme assays

• Quantitative Western blot analysis:

  • Use PFP-BETA1 antibody with chemiluminescent or fluorescent detection

  • Include recombinant PFP-BETA1 protein standards at known concentrations

  • Generate standard curves for absolute quantification

  • Normalize data to total protein or housekeeping proteins

• Enzyme activity measurements:

  • Assay PFP activity in both forward and reverse directions:

    • Forward: Fructose-6-P + PPi → Fructose-1,6-BP + Pi

    • Reverse: Fructose-1,6-BP + Pi → Fructose-6-P + PPi

  • Couple reactions to NAD(P)H oxidation/reduction for spectrophotometric detection

  • Include controls for substrate specificity (ATP vs. PPi dependence)

  • Perform kinetic analyses (Km, Vmax) in different tissues

• Correlation analysis:

  • Calculate specific activity (enzyme activity per unit of immunodetectable protein)

  • Generate correlation plots between protein levels and activity

  • Perform regression analysis to quantify relationships

  • Identify tissues with discrepancies suggesting post-translational regulation

• Validation experiments:

  • For tissues with discordant protein/activity ratios, investigate:

    • Post-translational modifications using phospho-specific antibodies

    • Protein-protein interactions using co-immunoprecipitation

    • Allosteric regulators using metabolite profiling

This integrated approach provides a comprehensive understanding of how PFP-BETA1 expression relates to functional activity across different tissues and conditions, potentially revealing tissue-specific regulatory mechanisms that fine-tune glycolytic flux .

How might researchers utilize PFP-BETA1 antibody to investigate potential non-glycolytic functions of this enzyme?

Recent advances in metabolic enzyme research have revealed that many glycolytic enzymes possess "moonlighting" functions beyond their canonical metabolic roles. Investigating potential non-glycolytic functions of PFP-BETA1 represents an exciting frontier in plant biology research. The following experimental approaches utilizing PFP-BETA1 antibody can help uncover such novel functions:

• Subcellular redistribution studies:

  • Track PFP-BETA1 localization under various stresses using immunofluorescence

  • Perform subcellular fractionation followed by Western blotting

  • Look for unexpected localizations (nucleus, membrane associations, organelles)

  • Correlate relocalization with specific cellular conditions

• Protein interactome mapping:

  • Perform immunoprecipitation with PFP-BETA1 antibody under diverse conditions

  • Identify interaction partners using mass spectrometry

  • Focus on non-glycolytic partners suggesting alternative functions

  • Validate interactions using reciprocal co-IPs and proximity ligation assays

• Post-translational modification profiling:

  • Use PFP-BETA1 antibody for enrichment prior to MS analysis

  • Identify condition-specific modifications (phosphorylation, acetylation, etc.)

  • Correlate modifications with non-glycolytic cellular processes

  • Generate modification-specific antibodies for further studies

• Functional screening approaches:

  • Utilize PFP-BETA1 antibody in chromatin immunoprecipitation if nuclear localization is observed

  • Screen for RNA-binding capacity through CLIP (cross-linking immunoprecipitation)

  • Investigate potential structural roles through cytoskeletal co-localization studies

  • Assess involvement in stress granules or other non-membranous compartments

This multifaceted approach can potentially reveal unexpected roles of PFP-BETA1 in processes such as transcriptional regulation, signal transduction, or structural organization, significantly expanding our understanding of how metabolic enzymes contribute to cellular homeostasis beyond their catalytic functions .

What methodological considerations are important when developing quantitative assays using PFP-BETA1 antibody?

Developing robust quantitative assays using PFP-BETA1 antibody requires careful methodological considerations to ensure accuracy, precision, and biological relevance. Researchers should address the following critical factors:

• Antibody characterization for quantitative applications:

  • Determine linear dynamic range of detection

  • Establish limits of detection and quantification

  • Assess lot-to-lot variability and implement standardization procedures

  • Validate specificity across the concentration range of interest

• Sample preparation optimization:

  • Evaluate extraction buffer composition effects on quantitative recovery

  • Assess protein stability during storage and processing

  • Determine optimal sample dilutions to remain within linear range

  • Implement spike-recovery experiments to verify extraction efficiency

• Standard curve generation:

  • Produce and purify recombinant PFP-BETA1 protein

  • Characterize standard purity and activity

  • Create multi-point calibration curves covering physiological concentration ranges

  • Include matrix-matched standards to account for sample complexity

• Assay format selection and optimization:

  • For Western blot quantification:

    • Implement fluorescent secondary antibodies for wider linear range

    • Use internal loading controls for normalization

    • Employ image analysis software with appropriate background correction

  • For ELISA development:

    • Optimize antibody coating concentration and blocking conditions

    • Determine optimal sample and antibody incubation times and temperatures

    • Evaluate detection system sensitivity and dynamic range

    • Implement four-parameter logistic regression for standard curve fitting

• Validation protocol:

  • Assess precision through intra- and inter-assay coefficient of variation determinations

  • Evaluate accuracy through spike-recovery experiments

  • Determine analytical specificity through cross-reactivity studies

  • Verify biological relevance by comparing with orthogonal methods

By addressing these methodological considerations, researchers can develop robust quantitative assays using PFP-BETA1 antibody that provide reliable and meaningful data, enabling more sophisticated investigations into the role of this enzyme in plant metabolism and beyond .