DEGP15 Antibody

What is DEG15 and what is its biological function?

DEG15 is a peroxisomal Deg-protease with endopeptidase activity that acts as a peroxisomal processing peptidase in plants. It specifically cleaves substrates containing cysteine in the P1 and P2 positions . This enzyme is crucial for processing peroxisomal targeting signal 2 (PTS2)-containing proteins, which is an essential step in peroxisomal protein import and function. The protease is also known by alternative names such as At1g28320/F3H9_2 in Arabidopsis thaliana . DEG15 plays a vital role in plant metabolism by enabling proper targeting and functionality of peroxisomal proteins, which are involved in critical processes including fatty acid β-oxidation, photorespiration, and detoxification of reactive oxygen species.

What species reactivity has been confirmed for DEGP15 antibody?

The DEGP15 antibody has been experimentally confirmed to react with several plant species:

This broad reactivity makes the antibody a valuable tool for comparative studies across different plant species . No confirmed exceptions from predicted reactivity are currently known, suggesting high conservation of the epitope across plant species.

What applications is the DEGP15 antibody validated for?

The DEGP15 antibody has been primarily validated for Western blot (WB) applications with a recommended dilution of 1:500 . While its primary application is in Western blotting, researchers should note that optimization might be required for other immunological techniques such as immunohistochemistry or immunoprecipitation. The antibody's specificity for the DEG15 protein makes it particularly valuable for studying peroxisomal biogenesis and protein processing in plant systems.

What is the optimal protocol for Western blot using DEGP15 antibody?

For optimal Western blot results with DEGP15 antibody, follow this research-validated protocol:

• Sample Preparation:

  • Extract total protein from plant tissue using a buffer containing protease inhibitors

  • Quantify protein and load 20-30 μg per lane

• Gel Electrophoresis and Transfer:

  • Separate proteins on 10% SDS-PAGE (optimal for detecting the 76 kDa DEG15 protein)

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Antibody Incubation:

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

  • Incubate with DEGP15 antibody at 1:500 dilution overnight at 4°C

  • Wash 3x with TBST, 10 minutes each

  • Incubate with secondary antibody (anti-rabbit HRP) at 1:5000 for 1 hour

  • Wash 3x with TBST, 10 minutes each

• Detection:

  • Visualize using ECL detection reagents

  • Expected molecular weight: 76 kDa

This protocol is based on established research practices and should be optimized for specific experimental conditions.

How should the DEGP15 antibody be reconstituted and stored?

The DEGP15 antibody is typically supplied in lyophilized format. For proper reconstitution and storage:

• Reconstitution:

  • Add 100 μl of sterile water to the lyophilized antibody

  • Gently mix to ensure complete dissolution

  • Allow to stand at room temperature for 5 minutes

• Storage:

  • Store lyophilized antibody at -20°C until reconstitution

  • Once reconstituted, store at -20°C

  • Make small aliquots to avoid repeated freeze-thaw cycles

  • Before each use, briefly centrifuge tubes to collect all material

• Stability Considerations:

  • Reconstituted antibody remains stable for approximately 6 months at -20°C

  • Avoid more than 3 freeze-thaw cycles to maintain activity

  • Record date of reconstitution on each aliquot

Proper storage and handling significantly impact antibody performance and experimental reproducibility.

What controls should be included when using DEGP15 antibody?

For rigorous scientific validation, include the following controls when using DEGP15 antibody:

• Positive Controls:

  • Wild-type Arabidopsis thaliana leaf extracts

  • Known DEG15-expressing plant tissues

• Negative Controls:

  • DEG15 knockout mutant extracts (e.g., Arabidopsis deg15 T-DNA insertion lines)

  • Pre-immune serum at equivalent concentration

• Loading Controls:

  • Housekeeping proteins (e.g., actin, tubulin)

  • Total protein stain (e.g., Ponceau S)

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide derived from Arabidopsis thaliana DEG15 (At1g28320)

  • Secondary antibody-only control

Including these controls ensures experimental rigor and facilitates accurate interpretation of results, especially when working with new tissue types or experimental conditions.

How can DEGP15 antibody be used to study peroxisomal biogenesis?

The DEGP15 antibody serves as a powerful tool for investigating peroxisomal biogenesis through several advanced applications:

• Protein Processing Studies:

  • Track processing of PTS2-containing proteins by comparing molecular weights before and after DEG15 activity

  • Monitor changes in processing efficiency under various physiological conditions

• Co-localization Analysis:

  • Combined with other peroxisomal markers to study protein import pathways

  • Immunofluorescence microscopy to visualize peroxisomal distribution and morphology

• Interaction Studies:

  • Co-immunoprecipitation to identify DEG15 binding partners

  • Analyze processing complex formation through native gel electrophoresis

• Comparative Analyses:

  • Cross-species studies leveraging the antibody's reactivity with multiple plant species

  • Evolutionary conservation of peroxisomal processing mechanisms

These approaches have provided significant insights into the fundamental mechanisms of peroxisomal protein import and processing, as demonstrated in studies such as Schuhmann et al. (2008) that investigated DEG15's role in cleaving peroxisomal targeting signal 2-containing proteins in Arabidopsis .

What is known about DEG15's dual specificity in plants?

DEG15 demonstrates remarkable dual specificity in plant systems, a characteristic elucidated through antibody-based research:

• Substrate Specificity:

  • Primary specificity for substrates with cysteine in P1 and P2 positions

  • Secondary specificity for certain non-canonical substrates

• Compartmental Functionality:

  • Functions in both glyoxysomes and peroxisomes

  • Processes distinct substrate pools in each compartment

This dual specificity was rigorously investigated in the pioneering work by Helm et al. (2007), which utilized antibody detection to characterize the glyoxysomal/peroxisomal processing capabilities of DEG15 in higher plants . The study revealed that DEG15 can process proteins destined for both compartments despite their different metabolic functions, suggesting an evolutionary conservation of processing mechanisms.

How can DEGP15 antibody be used in plant stress response studies?

The DEGP15 antibody provides valuable insights into plant stress responses through several methodological approaches:

• Expression Level Analysis:

  • Western blot quantification of DEG15 levels under various abiotic stresses

  • Correlation between stress intensity and protease abundance

• Processing Efficiency Studies:

  • Assessment of PTS2 protein processing rates under stress conditions

  • Determination of rate-limiting steps in peroxisomal protein import during stress

• Subcellular Distribution:

  • Changes in peroxisome abundance and morphology during stress responses

  • Redistribution of DEG15 between cellular compartments

• Workflow Integration:

  • Combined proteomics and immunodetection approaches

  • Integration with transcriptomic data for multilevel analysis

These applications have revealed that peroxisomal function, including DEG15 activity, is modulated during various stress responses, particularly oxidative stress and drought conditions, highlighting the importance of peroxisomal protein processing in plant adaptation.

What are common causes of background when using DEGP15 antibody?

Researchers may encounter background issues when using DEGP15 antibody, which can be addressed through systematic troubleshooting:

• High Background Causes and Solutions:

• Specialized Approaches:

  • For plant samples with high phenolic content, add PVPP to extraction buffer

  • Pre-absorb antibody with acetone powder from non-target species

  • Perform protein extraction under conditions that minimize proteolysis

These troubleshooting steps have been validated in research settings and can significantly improve signal-to-noise ratio when working with the DEGP15 antibody.

How can specificity of DEGP15 antibody be validated?

Validation of DEGP15 antibody specificity requires a multi-faceted approach:

• Primary Validation Methods:

  • Western blot comparison between wild-type and deg15 knockout plants

  • Peptide competition assay using the immunogenic peptide

  • Immunoprecipitation followed by mass spectrometry identification

• Secondary Validation Methods:

  • Recombinant protein expression and detection

  • Correlation between transcript and protein levels

  • Detection of expected molecular weight shifts after processing

• Validation Metrics:

  • Single band at expected molecular weight (76 kDa)

  • Absence of band in knockout tissues

  • Reproducible detection across technical replicates

These validation approaches ensure experimental rigor and confidence in attributing observed signals to authentic DEG15 protein, rather than non-specific interactions or artifacts.

What might explain unexpected molecular weight variants when detecting DEG15?

When researchers observe unexpected molecular weight variants in DEG15 detection, several biological and technical explanations should be considered:

• Biological Explanations:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splicing variants

  • Proteolytic processing during sample preparation

  • Species-specific variants (differences between predicted 76 kDa and observed weight)

• Technical Considerations:

  • Anomalous migration due to protein structure or buffer conditions

  • Incomplete denaturation affecting electrophoretic mobility

  • Gel percentage affecting resolution of higher molecular weight proteins

  • Differences between theoretical and apparent molecular weight

• Resolution Strategies:

  • Use gradient gels for better resolution

  • Compare reducing and non-reducing conditions

  • Treat samples with specific deglycosylation enzymes

  • Perform 2D gel electrophoresis for complex samples

Understanding these variables helps researchers correctly interpret their results and distinguish genuine biological phenomena from technical artifacts.

How does DEG15 function compare to other plant proteases?

DEG15 belongs to the larger family of Deg proteases but possesses distinctive characteristics:

This comparative analysis underscores DEG15's unique role in plant cell biology and peroxisomal function, distinguishing it from other proteases that may share structural similarities but fulfill distinct cellular roles.

What are the differences between plant and animal peroxisomal processing proteases?

Significant differences exist between plant DEG15 and animal peroxisomal processing proteases:

• Structural Differences:

  • Plant DEG15: Serine protease of the Deg/HtrA family

  • Mammalian counterpart: TYSND1 (Trypsin domain-containing 1)

• Substrate Processing:

  • Plant DEG15: Processes PTS2-containing proteins with specificity for Cys in P1/P2

  • Mammalian TYSND1: Processes both PTS1 and PTS2 proteins with broader specificity

• Evolutionary Implications:

  • Independent evolution of processing mechanisms

  • Convergent evolution of peroxisomal import systems

• Research Applications:

  • Plant DEG15 studies provide comparative insights into basic peroxisomal biology

  • Cross-kingdom comparative analyses reveal fundamental aspects of organelle evolution

This comparative understanding helps researchers place DEG15 function in a broader evolutionary context and highlights the unique aspects of plant peroxisomal processing.