PECR Antibody

What is PECR and what cellular functions does it perform?

PECR (peroxisomal trans-2-enoyl-CoA reductase) is an enzyme that participates in carbon chain elongation in fatty acid metabolism. It specifically catalyzes the last reaction of four long chain fatty acid elongation cycles . PECR is located on chromosome q35 with 86627 bases and is also known by alternative names such as TERP and TECR . The protein has a calculated molecular weight of 33 kDa, which corresponds to its observed molecular weight in experimental conditions .

In which tissues and cell types is PECR protein expressed?

Based on antibody validation data, PECR protein expression has been detected in multiple human tissues including testis, kidney, and liver cancer tissue . In addition, it has been successfully detected in HepG2 cells (human liver cancer cell line) . In mouse models, PECR has been validated in liver tissue using both Western blot and immunoprecipitation techniques . The expression pattern suggests that PECR plays important roles in metabolic processes across multiple organ systems.

What applications are PECR antibodies validated for?

PECR antibodies have been validated for multiple experimental applications:

The optimal dilution may be sample-dependent, and researchers should titrate the antibody in their specific testing systems to obtain optimal results .

How should PECR antibodies be stored for maximum stability?

For long-term storage, PECR antibodies should be stored at -20°C where they remain stable for one year after shipment . For short-term storage and frequent use, store at 4°C for up to one month . It is advisable to avoid repeated freeze-thaw cycles as this can degrade antibody quality and performance . Commercial preparations typically come in liquid form in PBS containing 50% glycerol and 0.02% sodium azide as preservatives . For antibodies in 20μL sizes, these often contain 0.1% BSA as a stabilizer .

What is the clinical relevance of anti-PECR antibodies in transplantation medicine?

Anti-PECR antibodies have emerging significance in transplantation medicine, particularly in kidney and lung transplants. Studies have demonstrated that the presence of anti-PECR antibodies strongly correlates with transplant glomerulopathy (TG), although this correlation is not associated with the pathologic grade of TG . Importantly, antibodies against PECR have been associated with both acute and chronic antibody-mediated rejection (AMR), and this association occurs independently of donor-specific antibodies (DSAs) .

In lung transplantation specifically, anti-PECR antibodies show strong correlation with chronic lung allograft dysfunction (CLAD) occurrence . These findings suggest that monitoring for anti-PECR antibodies in transplant recipients may provide valuable prognostic information about potential rejection events, particularly in cases where traditional markers like DSAs are not present.

What validation strategies should be employed to confirm PECR antibody specificity?

Validating PECR antibody specificity requires a multi-faceted approach:

• Knockout/Knockdown Validation: The gold standard for antibody validation includes testing in samples where PECR has been knocked out or knocked down. Published KD/KO data is available for certain PECR antibodies and should be consulted when selecting reagents .

• Multiple Detection Methods: Validate the antibody using more than one technique (e.g., WB, IHC, IP) to ensure consistent target recognition across different protein conformations and experimental conditions .

• Positive and Negative Controls: Always include appropriate tissue or cell samples known to express PECR (positive controls) and those known not to express it (negative controls) .

• Full Blot Analysis: When examining Western blot results, evaluate the entire blot rather than just the region of expected molecular weight to detect potential cross-reactivity or non-specific binding .

• Immunogen Sequence Analysis: Review the immunogen sequence used to generate the antibody (e.g., amino acids 40-120 of human PECR protein) to predict potential cross-reactivity with related proteins .

How can researchers optimize PECR antibody protocols for challenging samples or applications?

When working with challenging samples or applications, consider these optimization strategies:

• Antigen Retrieval Methodologies: For IHC applications with PECR antibodies, data suggests trying TE buffer pH 9.0 for antigen retrieval. Alternatively, citrate buffer pH 6.0 may be used if the primary method is ineffective .

• Protocol Adherence: Strictly follow manufacturer's protocols before introducing experimental variations. This establishes a baseline performance level before troubleshooting .

• Application-Specific Optimization: Even when an antibody is validated for multiple applications, each application may require different optimization parameters:

  • For WB: Adjust blocking reagents, incubation times, and washing stringency

  • For IHC: Test different fixatives, embedding methods, and counterstains

  • For IP: Modify lysis conditions, bead types, and elution methods

• Sample-Dependent Titration: The optimal antibody concentration may vary based on the sample type. Perform a titration series using samples similar to your experimental samples .

What criteria should guide PECR antibody selection for specific applications?

When selecting PECR antibodies for specific applications, consider the following criteria:

• Application-Specific Validation: Ensure the antibody has been directly tested and validated in your intended application (WB, IHC, IP, or ELISA) .

• Species Reactivity: Verify that the antibody has demonstrated reactivity with your species of interest. PECR antibodies may vary in their cross-reactivity between human, mouse, and rat samples .

• Clonality Considerations: Polyclonal antibodies like those described in the search results may offer broader epitope recognition but potentially more batch-to-batch variation compared to monoclonals .

• Immunogen Information: Review the immunogen sequence used to generate the antibody. For example, some PECR antibodies are raised against synthesized peptides derived from the human protein at amino acid range 40-120 . This helps predict which protein domains the antibody will recognize.

• Published Citations: Examine peer-reviewed publications that have successfully used the antibody in similar experimental contexts .

• Manufacturer Reputation: Source antibodies from reputable vendors with transparent validation practices and comprehensive technical support .

What are the recommended protocols for Western blot detection of PECR?

For optimal Western blot detection of PECR:

• Sample Preparation:

  • PECR has been successfully detected in HepG2 cells, human testis tissue, human kidney tissue, and mouse liver tissue .

  • Use appropriate lysis buffers that preserve protein integrity while solubilizing membrane-associated proteins.

• Loading Controls:

  • Include appropriate loading controls relevant to the subcellular fraction being examined.

  • Since PECR is peroxisomal, consider peroxisomal marker proteins as fraction-specific controls.

• Antibody Dilution:

  • Use PECR antibodies at the recommended dilution range of 1:2000-1:12000 for Western blot applications .

  • Titrate to determine the optimal concentration for your specific samples.

• Detection Method:

  • PECR has an observed molecular weight of 33 kDa, consistent with its calculated molecular weight .

  • Ensure your detection system has appropriate sensitivity in this molecular weight range.

• Validation Controls:

  • Include positive control samples with known PECR expression.

  • Where available, include knockdown or knockout samples as negative controls.

How can researchers troubleshoot inconsistent PECR antibody results?

When faced with inconsistent results using PECR antibodies:

• Antibody Validation Assessment:

  • Verify that the antibody has been properly validated for your specific application and sample type.

  • Check if published validation data includes knockdown/knockout controls .

• Technical Considerations:

  • Ensure proper storage and handling of the antibody (avoid repeated freeze-thaw cycles).

  • Verify buffer compatibility with your experimental conditions.

  • Check antibody age and stability; older antibodies may show decreased performance.

• Sample Preparation Variables:

  • Assess if differences in sample preparation might affect epitope accessibility.

  • For IHC applications, try alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) .

• Protocol Adherence:

  • Strictly follow the manufacturer's recommended protocols before introducing modifications .

  • Document all deviations from standard protocols to identify potential sources of variability.

• Blocking and Background Issues:

  • Optimize blocking conditions to reduce non-specific binding.

  • Adjust antibody concentration to improve signal-to-noise ratio.

What approaches can be used to quantify PECR expression levels across different samples?

For quantitative analysis of PECR expression:

• Western Blot Quantification:

  • Use digital imaging systems with appropriate dynamic range.

  • Normalize PECR signal to loading controls.

  • Generate standard curves using recombinant PECR protein if absolute quantification is needed.

• Immunohistochemistry Quantification:

  • Employ digital pathology software for objective scoring.

  • Use standardized scoring systems (H-score, Allred score, etc.) for semi-quantitative assessment.

  • Include reference standards on each slide for inter-slide normalization.

• ELISA-Based Quantification:

  • Develop quantitative ELISA protocols using purified PECR standards.

  • Validate assay linearity, sensitivity, and reproducibility.

• Multiple Antibody Approach:

  • When possible, use multiple antibodies targeting different PECR epitopes to confirm expression patterns.

  • Compare results across different detection methodologies (e.g., antibody-based vs. mRNA-based).

How can PECR antibodies be utilized in studies of fatty acid metabolism disorders?

Given PECR's role in fatty acid metabolism, antibodies against this protein can be valuable tools in studying related disorders:

• Peroxisomal Disorder Studies:

  • Use immunolocalization with PECR antibodies to assess peroxisome integrity and distribution in patient samples.

  • Combine with other peroxisomal markers to evaluate compartment-specific defects.

• Metabolic Disease Research:

  • Compare PECR expression and localization in normal vs. diseased liver tissues.

  • Correlate PECR levels with clinical parameters of fatty acid metabolism.

• Drug Development Applications:

  • Screen compounds that modify PECR expression or activity as potential therapeutics for lipid metabolism disorders.

  • Use PECR antibodies to assess drug effects on peroxisomal fatty acid processing pathways.

What insights can multiplex immunostaining with PECR antibodies provide?

Multiplexed approaches combining PECR with other markers can yield deeper insights:

• Subcellular Colocalization Studies:

  • Combine PECR antibodies with other peroxisomal markers to study organelle dynamics.

  • Assess potential interactions with mitochondrial or endoplasmic reticulum proteins involved in lipid metabolism.

• Tissue Context Analysis:

  • Use PECR alongside cell-type-specific markers to identify expression patterns within heterogeneous tissues.

  • Map PECR expression in relation to pathological features in disease models.

• Signaling Pathway Integration:

  • Combine with phospho-specific antibodies to correlate PECR expression with activation of relevant signaling cascades.

  • Study temporal relationships between PECR expression and metabolic stress responses.

What emerging technologies might enhance PECR antibody applications in research?

Several technological advances may enhance PECR antibody applications:

• Single-Cell Analysis:

  • Adaptation of PECR antibodies for mass cytometry or imaging mass cytometry to study expression at single-cell resolution.

  • Integration with single-cell transcriptomics to correlate protein and mRNA levels.

• Super-Resolution Microscopy:

  • Use of PECR antibodies with techniques like STORM or STED to visualize peroxisomal substructures.

  • Study dynamics of PECR localization with nanometer precision.

• Proximity Labeling Approaches:

  • Combine PECR antibodies with proximity labeling technologies to identify protein interaction networks.

  • Map the peroxisomal interactome in different metabolic states.

How might PECR antibodies contribute to biomarker development in transplantation medicine?

The correlation between anti-PECR antibodies and transplantation outcomes suggests potential biomarker applications:

• Non-invasive Monitoring:

  • Development of sensitive assays to detect anti-PECR antibodies in patient serum or plasma.

  • Longitudinal monitoring of antibody titers to predict rejection events.

• Multiparameter Risk Assessment:

  • Integration of anti-PECR antibody detection with other non-HLA antibody measurements.

  • Creation of risk algorithms that incorporate multiple antibody specificities.

• Therapeutic Response Prediction:

  • Use anti-PECR antibody levels to monitor response to desensitization therapies.

  • Correlate titer changes with clinical outcomes to guide treatment decisions.