P4H4 Antibody

What is P4H4 and what is its biological significance in plant research?

P4H4 (Probable prolyl 4-hydroxylase 4) is an enzyme that catalyzes the post-translational formation of 4-hydroxyproline in proline-rich peptide sequences of plant glycoproteins and other proteins. The enzyme specifically targets -Xaa-Pro-Gly- sequences. These hydroxyprolines are essential components of many plant cell wall glycoproteins such as extensins, hydroxyproline-rich glycoproteins, lectins, and arabinogalactan proteins. P4H4 is localized to the endoplasmic reticulum membrane and functions as a single-pass type II membrane protein. The gene is identified as At5g18900 in Arabidopsis thaliana, making it an important target in plant molecular biology research focused on cell wall formation and protein modification pathways.

What experimental applications is P4H4 Antibody validated for?

According to available research data, P4H4 Antibody has been validated for several key experimental applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB) for protein identification

These applications are particularly useful in plant molecular biology research focused on protein expression and modification studies. When designing experiments, researchers should note that the antibody has been specifically tested with Arabidopsis thaliana samples . The antibody is produced through antigen affinity purification methods, which ensures specificity for experimental applications .

What are the optimal storage and handling conditions for P4H4 Antibody?

For maintaining optimal antibody activity and stability, P4H4 Antibody should be stored at -20°C or -80°C immediately upon receipt. Multiple freeze-thaw cycles should be avoided as they can significantly reduce antibody effectiveness and specificity . The antibody is typically supplied in a liquid form with a storage buffer composition of:

• 0.03% Proclin 300 (preservative)

• 50% Glycerol

• 0.01M PBS at pH 7.4

This formulation helps maintain antibody stability during storage periods. For working dilutions, aliquoting the stock solution into smaller volumes is recommended to prevent repeated freeze-thaw cycles. Proper handling techniques include using sterile pipette tips and microcentrifuge tubes when preparing dilutions.

How should specificity and cross-reactivity be evaluated for P4H4 Antibody?

Evaluating the specificity of P4H4 Antibody requires several validation steps:

• Positive control testing: Using known P4H4-expressing Arabidopsis thaliana tissue or recombinant P4H4 protein.

• Negative control testing: Testing the antibody against tissues where P4H4 expression is knocked down or absent.

• Cross-reactivity assessment: Testing against related prolyl 4-hydroxylase family members to ensure specificity.

• Western blot validation: Confirming single band detection at the expected molecular weight.

The antibody has been raised against recombinant Arabidopsis thaliana P4H4 protein , which serves as its immunogen. This specific production method enhances its target recognition capabilities. When evaluating experimental results, researchers should be aware that P4H4 belongs to the P4HA family, and proper controls should be implemented to avoid misinterpreting signals from related family members.

How can P4H4 Antibody be employed to study plant stress responses and cell wall modifications?

P4H4 Antibody can serve as a powerful tool for investigating plant stress responses through several methodological approaches:

• Temporal expression analysis: Track P4H4 protein expression levels during exposure to various stressors (drought, salt, pathogens) using western blotting with the P4H4 Antibody.

• Spatial localization studies: Employ immunohistochemistry or immunofluorescence techniques to visualize changes in P4H4 distribution throughout plant tissues under stress conditions.

• Co-localization experiments: Combine P4H4 Antibody with markers for ER stress or cell wall integrity to determine relationships between P4H4 activity and cellular stress responses.

Since P4H4 catalyzes the formation of hydroxyproline in cell wall glycoproteins, monitoring its expression and activity can provide insights into how plants modify their cell walls in response to environmental challenges. This approach is particularly valuable for researchers investigating drought tolerance, pathogen resistance, or mechanical stress adaptations in plants.

What methodological approaches can overcome challenges in detecting low-abundance P4H4 in plant tissues?

Detecting low-abundance P4H4 protein in plant tissues presents several challenges that can be addressed through these methodological refinements:

• Sample enrichment protocols:

  • Subcellular fractionation focusing on the ER membrane fraction where P4H4 is localized

  • Immunoprecipitation to concentrate the target protein before detection

  • Optimized protein extraction buffers containing appropriate detergents for membrane protein solubilization

• Signal amplification strategies:

  • Enhanced chemiluminescence (ECL) detection systems with extended exposure times

  • Tyramine signal amplification for immunohistochemistry applications

  • Polymer-based detection systems rather than traditional secondary antibodies

• Validated loading controls: Implement consistent loading controls specific to the ER membrane compartment rather than general housekeeping proteins.

When working with developmental stages or tissues with potential low P4H4 expression, increasing the starting material quantity while maintaining the same final sample volume can significantly improve detection sensitivity.

How can P4H4 Antibody be utilized in comparative studies across different plant species?

Using P4H4 Antibody for cross-species studies requires careful consideration of several factors:

• Sequence homology analysis: Before experimental design, researchers should perform bioinformatics analysis of P4H4 protein sequence conservation across target species, focusing on the antibody's epitope region.

• Cross-reactivity validation protocol:

  • Western blot analysis using protein extracts from multiple species

  • Dilution series testing to determine optimal antibody concentration for each species

  • Peptide competition assays to confirm specificity in each new species

• Data normalization strategies:

  • Implement species-specific loading controls

  • Consider relative quantification rather than absolute values when comparing across species

  • Account for potential differences in extraction efficiency between species

What are the considerations for using P4H4 Antibody in protein-protein interaction studies?

When employing P4H4 Antibody for protein-protein interaction investigations, researchers should consider the following methodological approaches:

• Co-immunoprecipitation protocol optimization:

  • Determine optimal lysis conditions that preserve protein-protein interactions while effectively solubilizing membrane-bound P4H4

  • Test various detergent concentrations (0.1-1% NP-40, Triton X-100, or digitonin)

  • Include protease inhibitors and phosphatase inhibitors to prevent degradation during lengthy procedures

• Proximity ligation assay (PLA) implementation:

  • Use P4H4 Antibody in combination with antibodies against suspected interaction partners

  • Optimize fixation protocols to preserve ER membrane structure where P4H4 is localized

  • Include appropriate controls for antibody specificity and background signal

• Data validation approaches:

  • Confirm interactions through reciprocal co-IP experiments

  • Validate findings using orthogonal methods (yeast two-hybrid, split-GFP)

  • Perform competition assays with recombinant proteins

Given that P4H4 is an ER membrane protein, special attention must be paid to extraction conditions that effectively solubilize the protein while maintaining its native interactions. Cross-linking approaches prior to immunoprecipitation may help capture transient interactions that might otherwise be lost during purification steps.

What are the recommended dilution factors and incubation conditions for different applications?

Optimal working dilutions for P4H4 Antibody vary by application and should be determined empirically for each experimental system:

These ranges serve as starting points and should be optimized based on signal intensity and background levels in each specific experimental context. The antibody is supplied at a concentration that facilitates flexible dilution preparation .

What sample preparation techniques yield optimal results for P4H4 detection in plant tissues?

Effective sample preparation is critical for successful P4H4 detection in plant tissues:

• Tissue homogenization protocol:

  • Flash-freeze tissue samples in liquid nitrogen

  • Grind thoroughly to a fine powder using pre-chilled mortar and pestle

  • Maintain cold temperatures throughout the process to prevent protein degradation

• Extraction buffer composition:

  • Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl

  • Detergents: 1% Triton X-100 or 0.5% sodium deoxycholate

  • Protease inhibitors: PMSF (1 mM), complete protease inhibitor cocktail

  • Reducing agents: 5 mM DTT or 14 mM β-mercaptoethanol

• Membrane protein enrichment steps:

  • Perform differential centrifugation to isolate membrane fractions

  • Consider using specific ER membrane isolation protocols for enhanced P4H4 detection

  • Solubilize membrane proteins with appropriate detergent concentrations

Since P4H4 is localized to the endoplasmic reticulum membrane, targeted extraction protocols that focus on membrane protein isolation will likely yield better results than general protein extraction methods. Adding glycerol (10-15%) to extraction buffers can help stabilize the protein during purification.

What controls should be implemented when using P4H4 Antibody in immunological applications?

Implementing appropriate controls is essential for reliable and interpretable results:

• Positive controls:

  • Arabidopsis thaliana tissue samples with known P4H4 expression

  • Recombinant P4H4 protein (if available) as a reference standard

  • Previously validated positive samples

• Negative controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Pre-immune serum at equivalent dilution to primary antibody

  • P4H4 knockdown or knockout tissues/cells (if available)

  • Non-expressing tissues as biological negative controls

• Specificity controls:

  • Peptide competition assay using the immunizing antigen

  • Antibody dilution series to demonstrate concentration-dependent signal

  • Multiple antibody approach using different P4H4 antibodies (if available)

• Loading and transfer controls:

  • Total protein staining (Ponceau S, SYPRO Ruby)

  • ER-resident protein markers (to confirm efficient extraction of compartment-specific proteins)

  • Sample processing controls processed identically across experimental groups

These controls help distinguish specific P4H4 signal from background or non-specific interactions, particularly important given the rabbit polyclonal nature of the P4H4 Antibody .

How can P4H4 Antibody be integrated with other techniques for comprehensive protein analysis?

Integrating P4H4 Antibody with complementary techniques creates powerful research approaches:

• Mass spectrometry integration:

  • Immunoprecipitation with P4H4 Antibody followed by LC-MS/MS analysis of co-precipitated proteins

  • Identification of post-translational modifications on P4H4 protein

  • Quantitative proteomics to assess P4H4-associated protein complex changes under different conditions

• Microscopy techniques:

  • Super-resolution microscopy with P4H4 Antibody to visualize precise subcellular localization

  • Live-cell imaging using P4H4 Antibody fragments conjugated to quantum dots

  • Correlative light and electron microscopy for ultrastructural context

• Functional genomics approaches:

  • Combine P4H4 Antibody detection with gene expression analysis

  • Integrate with CRISPR-Cas9 gene editing for functional validation

  • Pair with RNA-seq data to correlate protein levels with transcript abundance

• Structural biology connections:

  • Use P4H4 Antibody for protein purification prior to structural studies

  • Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study conformational dynamics

  • Employ antibody epitope mapping to gain insights into protein structure

This multidisciplinary approach leverages the specificity of the P4H4 Antibody while overcoming the limitations of any single technique, providing researchers with comprehensive understanding of P4H4 biology in plant systems.

What are common causes of false negative results with P4H4 Antibody and how can they be addressed?

Several factors can contribute to false negative results when using P4H4 Antibody:

• Protein degradation issues:

  • Ensure complete protease inhibitor cocktails are used during sample preparation

  • Maintain cold temperatures throughout processing

  • Consider adding specific inhibitors for plant proteases (e.g., phenylmethylsulfonyl fluoride, leupeptin)

  • Avoid repeated freeze-thaw cycles of samples

• Inefficient protein extraction:

  • Optimize detergent type and concentration for membrane protein solubilization

  • Extend extraction time for tough plant tissues

  • Consider alternative homogenization methods (bead beating, sonication)

  • Use specialized membrane protein extraction kits

• Epitope masking problems:

  • Test different fixation protocols if applicable (adjust fixative type, concentration, and duration)

  • Incorporate antigen retrieval steps if necessary

  • Consider native versus denaturing conditions for protein detection

• Detection sensitivity limitations:

  • Employ signal amplification systems

  • Increase antibody concentration or incubation time

  • Reduce washing stringency while monitoring background

  • Use higher sensitivity detection substrates

When addressing potential false negatives, systematic optimization of each experimental step while maintaining appropriate controls is the most effective approach.

How can batch-to-batch variation in polyclonal P4H4 Antibody be managed in long-term research projects?

Managing antibody variation is crucial for research reproducibility:

• Initial validation protocol:

  • Validate each new antibody lot against a standard sample set

  • Determine optimal working dilutions for each lot

  • Document lot-specific performance characteristics

• Reference standard implementation:

  • Maintain a stable reference sample set for cross-lot comparisons

  • Consider preparing a large batch of positive control lysate, aliquoting and storing at -80°C

  • Generate calibration curves for each lot if quantitative comparisons are needed

• Experimental design considerations:

  • Complete key comparative experiments with a single antibody lot when possible

  • Include internal reference samples across experiments using different lots

  • Develop normalization strategies to account for sensitivity differences between lots

• Alternative approaches for critical experiments:

  • Consider generating monoclonal antibodies for highly sensitive applications

  • Explore recombinant antibody technology for consistent renewable reagents

  • Implement orthogonal detection methods to validate key findings

Since P4H4 Antibody is polyclonal , these strategies are particularly important for maintaining consistency across extended research timelines.

How should quantitative data from P4H4 Antibody experiments be normalized and statistically analyzed?

• Normalization strategies:

  • Total protein normalization using stain-free technology or total protein stains

  • Housekeeping protein normalization using ER-specific markers (more relevant than general housekeeping proteins)

  • Relative quantification against internal reference samples

• Statistical analysis approaches:

  • Perform at least three biological replicates for each experimental condition

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Consider ANOVA with post-hoc tests for multiple group comparisons

  • Implement linear mixed models for complex experimental designs

• Data presentation guidelines:

  • Present both raw and normalized data when possible

  • Provide clear descriptions of normalization methods

  • Include error bars representing standard deviation or standard error

  • Report exact p-values rather than significance thresholds

When analyzing P4H4 expression across different experimental conditions, always consider the biological context, including potential diurnal variations in expression, developmental stage differences, and stress-induced changes that might influence baseline levels.

What biological insights can be derived from P4H4 localization and expression pattern studies?

P4H4 localization and expression pattern studies can yield valuable biological insights:

• Developmental regulation understanding:

  • Track P4H4 expression across developmental stages to identify critical periods of cell wall formation

  • Correlate P4H4 patterns with tissue differentiation events

  • Examine potential roles in specialized structures requiring modified cell walls

• Stress response mechanisms:

  • Analyze P4H4 expression changes during abiotic stresses (drought, salt, temperature)

  • Investigate alterations during pathogen challenge and immune responses

  • Determine relationship between P4H4 activity and mechanical stress adaptations

• Subcellular dynamics interpretations:

  • Evaluate P4H4 distribution within the ER membrane under different conditions

  • Identify potential relationships with other ER-resident enzymes

  • Assess trafficking patterns during high secretory activity periods

• Evolutionary conservation analysis:

  • Compare P4H4 expression patterns across plant species

  • Identify conserved vs. species-specific regulation

  • Connect expression differences to ecological adaptations

These studies can contribute to broader understanding of cell wall development, stress adaptation mechanisms, and the evolutionary significance of protein hydroxylation in plant biology.