DEGP9 Antibody

What is DEGP9 protein and what role does it play in Arabidopsis thaliana physiology?

DEGP9 (Q9FL12) is a serine protease belonging to the DEG/HtrA family in Arabidopsis thaliana. This protein functions primarily in protein quality control and stress response mechanisms within plant cells. As a protease, it participates in the degradation of misfolded or damaged proteins, particularly under stress conditions such as heat, oxidative stress, or pathogen attack. The proper functioning of DEGP9 is essential for maintaining cellular proteostasis and contributing to the plant's ability to withstand environmental challenges. Research into DEGP9 provides valuable insights into fundamental plant stress response mechanisms and protein quality control systems that can potentially inform agricultural applications related to crop resilience .

What are the optimal storage and handling conditions for DEGP9 Antibody?

For maintaining DEGP9 Antibody functionality, store upon receipt at either -20°C or -80°C according to the manufacturer's recommendations. Avoid repeated freeze-thaw cycles as these can significantly degrade antibody quality and diminish immunoreactivity. The antibody is supplied in a stabilizing liquid formulation containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. When handling, always use sterile technique and wear appropriate personal protective equipment. For short-term use during experiments, maintain the antibody on ice. Prior to use, centrifuge briefly to collect the solution at the bottom of the tube. For dilutions, use fresh, cold buffer solutions and prepare only the amount needed for immediate use to maintain optimal antibody performance .

How should DEGP9 Antibody be validated before experimental use?

Validation of DEGP9 Antibody requires a multi-step approach to ensure specificity and functionality. Begin with Western blot analysis using both wild-type Arabidopsis thaliana tissue extracts and degp9 knockout/knockdown mutant samples as a negative control. Confirm a single band of appropriate molecular weight (approximately 40-45 kDa for DEGP9) in wild-type samples that is absent or significantly reduced in mutant samples. Additionally, perform preabsorption tests by incubating the antibody with excess purified DEGP9 recombinant protein before immunostaining. Successful preabsorption should eliminate or substantially reduce signal detection. For further validation, consider peptide competition assays and testing cross-reactivity with other DEGP family members (particularly DEGP2, DEGP7, and DEG15) to confirm specificity. Document all validation steps thoroughly, including positive and negative controls, to establish confidence in subsequent experimental applications .

What is the recommended protocol for using DEGP9 Antibody in Western blotting with Arabidopsis samples?

For Western blotting with DEGP9 Antibody, begin by extracting total protein from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and protease inhibitor cocktail. Homogenize tissue thoroughly and centrifuge at 14,000 × g for 15 minutes at 4°C. Separate proteins (20-30 μg) on a 10-12% SDS-PAGE gel and transfer to a PVDF membrane. Block the membrane with 5% non-fat milk in TBST for 1 hour at room temperature. Incubate with DEGP9 Antibody at 1:1000 dilution in blocking buffer overnight at 4°C. Wash three times with TBST (5 minutes each), then incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature. After washing, develop using chemiluminescent substrate. When analyzing chloroplast-localized DEGP9, consider additional subcellular fractionation steps to enrich for chloroplast proteins. Include both positive controls (wild-type plants) and negative controls (degp9 mutants) to validate specificity. The expected molecular weight for DEGP9 is approximately 40-45 kDa, though post-translational modifications may cause slight variations in migration pattern .

How can DEGP9 Antibody be optimized for ELISA applications?

For ELISA applications with DEGP9 Antibody, begin optimization using a standard indirect ELISA format. Coat high-binding 96-well plates with 50-100 μl of Arabidopsis protein extract (2-5 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T (PBS with 0.05% Tween-20), block with 3% BSA in PBS for 1-2 hours at room temperature. Test DEGP9 Antibody across a range of dilutions (1:500, 1:1000, 1:2000, 1:5000) in blocking buffer to determine optimal concentration, incubating for 2 hours at room temperature. Follow with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour and develop with TMB substrate. Perform titration experiments to identify the antibody concentration yielding the highest signal-to-noise ratio. Include plant extracts from degp9 knockout mutants as negative controls to assess specificity and cross-reactivity. For quantitative ELISA, develop a standard curve using purified recombinant DEGP9 protein at known concentrations. Optimize incubation times and temperatures to enhance sensitivity while maintaining specificity for research-quality results .

How can DEGP9 Antibody be used to investigate protein-protein interactions in stress response pathways?

To investigate protein-protein interactions involving DEGP9 using its specific antibody, implement co-immunoprecipitation (co-IP) assays optimized for plant tissues. Begin by extracting total protein from Arabidopsis under conditions that preserve protein complexes (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Triton X-100, protease inhibitors). Pre-clear lysates with Protein A/G beads for 1 hour at 4°C. Incubate 500-1000 μg of cleared lysate with 2-5 μg of DEGP9 Antibody overnight at 4°C with gentle rotation. Add fresh Protein A/G beads and incubate for 3-4 hours at 4°C. Wash beads 4-5 times with wash buffer containing reducing detergent concentrations. Elute protein complexes with SDS sample buffer and analyze by SDS-PAGE followed by Western blotting for potential interacting partners. For stress response studies, compare protein interactions under normal conditions versus various stressors (heat, oxidative, salt stress). Combine with mass spectrometry to identify novel interacting partners in an unbiased approach. Validate identified interactions using reciprocal co-IPs and in vitro binding assays to distinguish direct versus indirect interactions. Consider crosslinking approaches for transient or weak interactions that might not survive traditional co-IP protocols .

What methods can be used to quantify changes in DEGP9 expression and localization during different plant stress conditions?

To quantify DEGP9 expression and localization changes during stress conditions, employ a multi-faceted approach combining quantitative immunoblotting, immunofluorescence microscopy, and subcellular fractionation techniques. For quantitative Western blotting, subject Arabidopsis plants to various stressors (heat shock, drought, pathogen exposure) with appropriate time-course sampling. Extract proteins from treated and control plants, perform Western blotting with DEGP9 Antibody, and quantify band intensities using densitometry software. Normalize to loading controls like actin or GAPDH for relative quantification. For subcellular localization studies, perform cellular fractionation to isolate chloroplasts, mitochondria, and cytosolic fractions, followed by immunoblotting of each fraction. Alternatively, use confocal microscopy with immunofluorescence labeling using DEGP9 Antibody followed by fluorophore-conjugated secondary antibodies. Counterstain with organelle-specific markers to track stress-induced relocalization events. For enhanced quantification precision, combine with qRT-PCR to correlate protein levels with transcriptional changes. When analyzing results, employ statistical methods such as ANOVA with appropriate post-hoc tests to determine significant changes across treatment conditions. Present data in tabular form showing relative expression levels across different stress conditions and time points .

How can contradictory results with DEGP9 Antibody be resolved in experimental settings?

When faced with contradictory results using DEGP9 Antibody, implement a systematic troubleshooting approach. First, re-validate antibody specificity through Western blotting with both wild-type and degp9 mutant tissues to confirm the antibody recognizes the correct target. Second, evaluate potential cross-reactivity with other DEGP family members (DEGP2, DEGP7, DEG15) as these share structural similarities and may confound results. Third, examine buffer compositions and experimental conditions, as DEGP9 detection may be sensitive to pH, salt concentration, or detergent types. Fourth, consider epitope masking due to protein-protein interactions or post-translational modifications that may occur under specific experimental conditions. Fifth, assess antibody lot-to-lot variability by comparing results with different batches or sources of anti-DEGP9 antibodies. Sixth, investigate tissue-specific or developmental regulation that might explain seemingly contradictory results in different sample types. Prepare a detailed protocol standardization document recording all variables (antibody dilutions, incubation times/temperatures, buffer compositions) to ensure consistency. Finally, consider employing complementary approaches such as mass spectrometry, fluorescent protein tagging, or alternative antibodies targeting different epitopes to corroborate findings and resolve contradictions .

What experimental design is recommended for studying DEGP9 protein degradation activity in vitro?

For studying DEGP9 protein degradation activity in vitro, design an experimental approach that combines recombinant protein production with functional protease assays. First, express and purify full-length DEGP9 protein with appropriate tags (His or GST) that can be removed before activity assays. Second, prepare fluorogenic peptide substrates containing known protease recognition sequences linked to quenched fluorophores that emit signal upon cleavage. Alternatively, use well-characterized protein substrates such as β-casein or oxidized BSA. Third, establish optimal reaction conditions by testing different buffers (50 mM Tris-HCl, pH 7.5-8.5), temperatures (20-45°C), and cofactor requirements (divalent cations like Ca²⁺ or Mg²⁺). Fourth, design time-course experiments to determine reaction kinetics by sampling at regular intervals (0, 15, 30, 60, 120 minutes). Fifth, include appropriate controls: heat-inactivated DEGP9 (negative control), commercial serine proteases (positive control), and reactions with serine protease inhibitors (PMSF or chymostatin). Sixth, analyze reaction products using SDS-PAGE with silver staining or Western blotting with DEGP9 Antibody to detect both substrate degradation and potential DEGP9 auto-processing. For quantitative analysis, prepare a standard curve correlating substrate degradation with fluorescence intensity or densitometric measurements. Present results as specific activity (μmol substrate cleaved per minute per mg enzyme) under different experimental conditions .

How should experiments be designed to investigate DEGP9 function using both genetic and immunological approaches?

To comprehensively investigate DEGP9 function using complementary genetic and immunological approaches, implement a multi-tiered experimental design. Begin with genetic characterization using T-DNA insertion mutants, CRISPR/Cas9-generated knockouts, and RNAi lines with varying degrees of DEGP9 suppression. Verify knockout/knockdown efficiency through RT-PCR, qRT-PCR, and Western blotting with DEGP9 Antibody. Generate complementation lines expressing DEGP9 under native promoter in the knockout background to confirm phenotype rescue. For protein-level analysis, use DEGP9 Antibody in immunoprecipitation experiments followed by mass spectrometry to identify interacting partners and potential substrates. Perform comparative phenotypic analysis of wild-type and mutant plants under normal and stress conditions (heat, drought, high light), documenting morphological, physiological, and biochemical parameters. Incorporate tissue-specific expression studies using DEGP9 promoter-reporter fusions alongside immunohistochemistry with DEGP9 Antibody to correlate gene expression with protein localization. For functional redundancy assessment, generate double/triple mutants with related DEGP family members and analyze synthetic phenotypes. Implement quantitative proteomic approaches comparing wild-type and degp9 mutant proteomes to identify accumulating substrates. Design experiments with appropriate biological replicates (minimum n=3) and include statistical analysis (ANOVA with post-hoc tests) to determine significant differences between genotypes and treatment conditions .

What data analysis methods are most appropriate for quantifying DEGP9 protein levels across different plant developmental stages?

For rigorous quantification of DEGP9 protein levels across plant developmental stages, implement a comprehensive data analysis workflow that combines multiple normalization strategies with appropriate statistical methods. Begin by collecting Arabidopsis tissue samples at defined developmental stages (seedling, vegetative, bolting, flowering, silique formation, senescence) with at least 3-4 biological replicates per stage. Extract proteins using standardized methods and separate equal amounts by SDS-PAGE for Western blotting with DEGP9 Antibody. Include multiple loading controls (actin, GAPDH, RuBisCO large subunit) on each blot to account for tissue-specific variation in housekeeping proteins. Capture images using a digital imaging system with linear dynamic range and analyze band intensities using software like ImageJ or specialized Western blot quantification tools. For normalization, calculate the ratio of DEGP9 signal to each loading control separately, then assess consistency across different normalization standards. Apply ANOVA with Tukey's HSD post-hoc test to determine statistically significant differences between developmental stages. Present data in both tabular and graphical formats showing normalized DEGP9 expression levels with error bars representing standard deviation or standard error. Complement protein-level analysis with transcriptional data (qRT-PCR of DEGP9 mRNA) to identify potential post-transcriptional regulation. For enhanced rigor, consider absolute quantification using purified recombinant DEGP9 protein standards to generate a calibration curve relating band intensity to protein concentration .

How can DEGP9 Antibody be used to investigate post-translational modifications of the DEGP9 protein?

To investigate post-translational modifications (PTMs) of DEGP9 protein using its specific antibody, implement a comprehensive workflow combining immunoprecipitation with specialized analytical techniques. First, immunoprecipitate DEGP9 from Arabidopsis tissues using optimized conditions (buffer containing phosphatase and deubiquitinase inhibitors) to preserve labile modifications. Perform parallel IPs from plants subjected to different stress conditions to identify stress-induced PTMs. Analyze immunoprecipitated DEGP9 using multiple approaches: (1) Western blotting with modification-specific antibodies (anti-phospho, anti-ubiquitin, anti-SUMO) alongside DEGP9 Antibody; (2) Phos-tag SDS-PAGE to detect phosphorylated forms through mobility shift; (3) 2D-gel electrophoresis to separate DEGP9 isoforms by both isoelectric point and molecular weight. For comprehensive PTM mapping, subject immunoprecipitated DEGP9 to mass spectrometry analysis using techniques optimized for PTM detection (enrichment strategies for phosphopeptides, titanium dioxide chromatography). Compare PTM profiles between control and stress-treated samples to identify condition-specific modifications. Validate functional significance of identified PTMs through site-directed mutagenesis of modified residues in complementation constructs (phosphomimetic mutations) followed by phenotypic analysis. Present data in tabular format showing identified PTMs, their position in the protein sequence, the techniques used for detection, and their relative abundance under different experimental conditions .