CYP83A1 Antibody

What is CYP83A1 and why is it important in plant research?

CYP83A1 is a cytochrome P450 monooxygenase that plays a crucial role in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. It functions by catalyzing the initial conversion of aliphatic aldoximes to thiohydroximates in the glucosinolate biosynthetic pathway. Understanding CYP83A1 is important because it represents a key regulatory point between primary metabolism and specialized metabolite production that impacts plant defense responses. The enzyme has been shown to preferentially metabolize aliphatic oximes derived from chain-elongated homologs of methionine with high efficiency . CYP83A1 shares 63% amino acid sequence identity with its close relative CYP83B1, but they have distinct substrate preferences and physiological roles in planta, making them non-redundant enzymes under normal conditions .

How can CYP83A1 antibodies be used to study glucosinolate biosynthesis?

CYP83A1 antibodies provide valuable tools for investigating glucosinolate biosynthesis through multiple techniques:

• Protein detection and quantification: Western blotting with specific CYP83A1 antibodies allows researchers to monitor protein expression levels across different tissues, developmental stages, or in response to biotic/abiotic stresses.

• Immunolocalization: Antibodies enable subcellular and tissue-specific localization of CYP83A1, providing insights into the spatial organization of glucosinolate biosynthesis machinery.

• Protein complex analysis: Immunoprecipitation using CYP83A1 antibodies can help identify protein-protein interactions within the glucosinolate biosynthetic complex.

• Enzyme activity verification: Antibodies can confirm the presence and integrity of CYP83A1 in enzymatic assays where recombinant or native proteins are used to study substrate specificity .

What are the key differences between CYP83A1 and CYP83B1 that antibodies can help distinguish?

While CYP83A1 and CYP83B1 share 63% amino acid sequence identity, they show marked differences in substrate specificity that can be investigated using specific antibodies . CYP83A1 preferentially metabolizes aliphatic oximes derived from chain-elongated homologs of methionine, while CYP83B1 has higher affinity for aromatic oximes, particularly indole-3-acetaldoxime (with a 50-fold lower Km value compared to CYP83A1) .

The table below summarizes the substrate specificity differences that antibody-based studies can help elucidate:

Antibodies specific to unique epitopes of each enzyme allow researchers to distinguish between these closely related proteins in complex biological samples and determine their relative expression levels or localization patterns .

What are the recommended protocols for using CYP83A1 antibodies in Western blotting?

When implementing Western blotting protocols for CYP83A1 detection, researchers should consider the following methodological approach:

• Sample preparation: Extract total protein from plant tissues using a buffer containing detergents suitable for membrane proteins (CYP83A1 is a membrane-associated P450 enzyme). Include protease inhibitors to prevent degradation.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal resolution of CYP83A1 (molecular weight approximately 58 kDa).

• Transfer conditions: Semi-dry or wet transfer to PVDF membranes (preferred over nitrocellulose for P450 proteins) at 100V for 1 hour or 30V overnight at 4°C.

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute CYP83A1-specific antibodies 1:1000 to 1:5000 in blocking buffer and incubate overnight at 4°C with gentle agitation.

• Controls: Always include positive controls (e.g., recombinant CYP83A1) and negative controls (e.g., protein extracts from cyp83a1 knockout mutants like cyp83a1-1 or cyp83a1-2) .

• Cross-reactivity considerations: Due to the 63% sequence identity with CYP83B1, confirm antibody specificity using samples from knockout mutants of both genes to ensure selective detection .

How can immunolocalization with CYP83A1 antibodies reveal spatial patterns of glucosinolate biosynthesis?

Immunolocalization using CYP83A1 antibodies provides valuable insights into the spatial organization of glucosinolate biosynthesis at tissue and cellular levels:

• Tissue fixation: Fix plant tissues in 4% paraformaldehyde, followed by paraffin embedding or cryosectioning.

• Antigen retrieval: For paraffin sections, perform antigen retrieval using citrate buffer (pH 6.0) to expose epitopes potentially masked during fixation.

• Antibody incubation: Apply CYP83A1 primary antibodies (1:100-1:500 dilution) followed by fluorophore-conjugated secondary antibodies.

• Co-localization analysis: Perform dual immunolabeling with antibodies against other glucosinolate biosynthetic enzymes (e.g., CYP83B1, CYP79F1) to map the complete pathway organization.

• Confocal microscopy: Use confocal laser scanning microscopy for high-resolution imaging of subcellular localization.

• Interpretation considerations: When interpreting results, consider that CYP83A1 is likely associated with the endoplasmic reticulum membrane, like other P450 enzymes, and may show tissue-specific expression patterns related to glucosinolate accumulation sites .

What controls should be included when performing immunoprecipitation using CYP83A1 antibodies?

When conducting immunoprecipitation (IP) experiments with CYP83A1 antibodies, the following controls are essential for reliable interpretation:

• Input control: Analyze a fraction of the total protein extract before IP to confirm the presence of CYP83A1.

• Negative genetic control: Perform parallel IP using extracts from cyp83a1 knockout plants (e.g., cyp83a1-1, cyp83a1-2, or cyp83a1-3) to identify non-specific interactions .

• Isotype control: Use an irrelevant antibody of the same isotype to identify non-specific binding to the antibody class.

• Pre-immune serum control: For polyclonal antibodies, include the pre-immune serum as a negative control.

• Reciprocal IP: If investigating protein-protein interactions, confirm findings by performing reverse IP with antibodies against the putative interacting partner.

• Competitive peptide control: Pre-incubate the antibody with the peptide used for immunization to block specific binding sites.

• Cross-reactivity assessment: Due to the similarity with CYP83B1 (63% sequence identity), include recombinant CYP83B1 protein to evaluate potential cross-reactivity of the antibody .

How can CYP83A1 antibodies help investigate the impact of mutations on protein stability and function?

CYP83A1 antibodies are instrumental in characterizing how mutations affect protein stability, structure, and function through several approaches:

• Mutant protein expression analysis: Western blotting with CYP83A1 antibodies can detect changes in protein abundance in mutant lines, revealing whether mutations affect protein stability. This approach was valuable in characterizing cyp83a1 mutants, including cyp83a1-3 which contains a G346E substitution in the heme-binding site .

• Structural integrity assessment: Antibodies recognizing different epitopes can help determine if mutations alter protein folding or domain structure.

• Immunoprecipitation of variant proteins: CYP83A1 antibodies can be used to isolate mutant proteins for subsequent activity assays or structural analyses.

• Subcellular localization comparison: Immunolocalization can reveal if mutations affect proper targeting of CYP83A1 to the endoplasmic reticulum or cause protein mislocalization.

• Protein-protein interaction changes: Co-immunoprecipitation using CYP83A1 antibodies can identify altered interaction patterns between mutant CYP83A1 and other enzymes in the glucosinolate biosynthetic pathway.

Research on cyp83a1 mutants has shown that specific mutations can dramatically alter plant defense responses, with the cyp83a1-3 mutant exhibiting enhanced resistance to powdery mildew fungi like Golovinomyces cichoracearum, demonstrating the importance of proper CYP83A1 function in regulating plant immunity .

What approaches can be used to investigate CYP83A1 regulation during plant defense responses using antibodies?

Investigating CYP83A1 regulation during plant defense responses using antibodies can be approached through several sophisticated methodologies:

• Temporal expression profiling: Using Western blotting with CYP83A1 antibodies to track protein levels at multiple time points after pathogen challenge or defense elicitor treatment.

• Phosphorylation state analysis: Combining immunoprecipitation with CYP83A1 antibodies followed by phospho-specific antibody detection or mass spectrometry to identify post-translational modifications that may regulate enzyme activity during defense responses.

• Chromatin immunoprecipitation (ChIP): Using antibodies against transcription factors potentially regulating CYP83A1 to identify defense-related transcriptional regulators, particularly focusing on the differing cis-acting elements in CYP83A1 and CYP83B1 promoters .

• Co-regulatory network analysis: Comparing expression patterns of CYP83A1 with other defense-related proteins using multiplexed Western blotting.

• Translational regulation assessment: Combining polysome profiling with CYP83A1 antibody detection to investigate translational control during defense responses.

Studies on cyp83a1-3 mutants have revealed that CYP83A1 functions in a defense pathway requiring NPR1, EDS1, and PAD4, but interestingly, not SID2 or EDS5, providing critical insights into how this enzyme integrates with established plant defense signaling networks .

How can CYP83A1 antibodies be utilized in investigating the interplay between glucosinolate biosynthesis and auxin homeostasis?

CYP83A1 antibodies provide powerful tools for examining the complex interplay between glucosinolate biosynthesis and auxin homeostasis through several sophisticated approaches:

• Dual-pathway protein complex analysis: Using CYP83A1 antibodies for co-immunoprecipitation studies to identify protein complexes that might simultaneously regulate both glucosinolate biosynthesis and auxin pathways.

• Comparative expression studies: Quantitative Western blotting with antibodies against both CYP83A1 and CYP83B1 to examine their differential regulation across tissues and developmental stages, considering that CYP83B1 has evolved to selectively metabolize indole-3-acetaldoxime, which is a shared intermediate with auxin biosynthesis .

• Metabolic flux analysis combined with protein detection: Correlating CYP83A1 protein levels with metabolite profiles of both pathways through integrated proteomics and metabolomics approaches.

• Hormone response element interactions: Using ChIP with antibodies against auxin-responsive transcription factors to investigate their binding to the promoter regions of CYP83A1, particularly relevant since CYP83B1 (but not CYP83A1) promoters contain putative auxin responsive cis-acting elements .

• Protein-metabolite interaction studies: Using CYP83A1 antibodies in protein-metabolite co-precipitation experiments to investigate if auxin or its precursors directly interact with and potentially regulate CYP83A1.

Research has shown that knockout mutants of CYP83B1 (rnt1-1) display a strong auxin excess phenotype, whereas CYP83A1 mutants do not show this phenotype. Interestingly, ectopic overexpression of CYP83A1 using a 35S promoter rescues the rnt1-1 phenotype, indicating that CYP83A1 can metabolize indole-3-acetaldoxime when expressed at sufficiently high levels, despite having a 50-fold lower affinity for this substrate compared to CYP83B1 .

What are common challenges in Western blot detection of CYP83A1 and how can they be addressed?

Researchers working with CYP83A1 antibodies may encounter several technical challenges in Western blotting that can be addressed through specific modifications:

• Low signal intensity: CYP83A1 may be expressed at relatively low levels in some tissues. Solutions include:

  • Concentrate proteins using immunoprecipitation before Western blotting

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Increase sample loading (50-100 μg total protein)

  • Extend primary antibody incubation to overnight at 4°C

• Non-specific bands: The 63% sequence identity with CYP83B1 may cause cross-reactivity . Address this by:

  • Pre-adsorbing antibodies with recombinant CYP83B1 protein

  • Using cyp83a1 knockout tissue (e.g., cyp83a1-1, cyp83a1-2) as negative controls

  • Increasing blocking time and washing stringency

  • Utilizing monoclonal antibodies targeting unique epitopes of CYP83A1

• Inconsistent membrane transfer: P450 proteins can be challenging to transfer efficiently due to their hydrophobic domains. Optimize by:

  • Using PVDF membranes instead of nitrocellulose

  • Adding 0.1% SDS to the transfer buffer to improve protein mobilization

  • Extending transfer time or using graduated transfer methods

• Sample degradation: To preserve CYP83A1 integrity:

  • Include protease inhibitor cocktails in extraction buffers

  • Maintain samples at 4°C throughout preparation

  • Add reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Background noise: Reduce background by:

  • Using freshly prepared buffers

  • Increasing washing steps (at least 3 x 10 minutes with TBST)

  • Optimizing secondary antibody dilution (typically 1:5000-1:10000)

How can antibody-based approaches help resolve contradictory data in CYP83A1 function studies?

Antibody-based techniques offer powerful solutions for resolving contradictory findings in CYP83A1 research:

• Protein expression verification: When functional studies yield inconsistent results, use quantitative Western blotting with CYP83A1 antibodies to confirm protein expression levels across experimental systems, ensuring that phenotypic variations aren't simply due to differences in protein abundance.

• Characterization of splice variants or isoforms: Antibodies recognizing different epitopes can detect potential CYP83A1 isoforms that might explain functional differences observed across studies.

• Post-translational modification analysis: Immunoprecipitation with CYP83A1 antibodies followed by mass spectrometry can identify modifications (phosphorylation, glycosylation, etc.) that might alter enzyme activity in different experimental conditions.

• Native complex integrity assessment: Blue-native PAGE combined with CYP83A1 antibody detection can reveal whether the enzyme exists in different protein complexes that might explain functional variations.

• Subcellular localization comparison: Immunolocalization across different experimental systems can identify potential differences in CYP83A1 targeting that might explain contradictory findings.

This approach proved valuable in understanding why CYP83A1 expression under its endogenous promoter in the rnt1-1 (CYP83B1 knockout) background failed to rescue the indole glucosinolate deficit, while 35S promoter-driven expression did rescue the phenotype—suggesting that expression level and pattern are critical factors in determining functional redundancy between these enzymes .

What strategies can optimize immunoprecipitation efficiency for low-abundance CYP83A1 protein?

Optimizing immunoprecipitation for low-abundance CYP83A1 requires specialized approaches:

• Crosslinking optimization: Implement a two-step crosslinking approach:

  • First, use membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate])

  • Follow with formaldehyde fixation (0.5-1%)

  • Carefully optimize crosslinking time (typically 10-30 minutes) to balance complex preservation with epitope accessibility

• Extraction buffer enhancement:

  • Include 0.5-1% digitonin or 1% Triton X-100 for efficient membrane protein solubilization

  • Add 10% glycerol to stabilize protein structure

  • Include phosphatase inhibitors if studying phosphorylation state

  • Consider using plant-specific extraction buffers containing polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

• Antibody immobilization strategies:

  • Covalently couple CYP83A1 antibodies to magnetic beads to prevent antibody leaching

  • Use oriented coupling methods to maximize antigen-binding capacity

  • Consider using nanobodies or camelid single-domain antibodies for improved access to conformational epitopes

• Signal amplification techniques:

  • Implement tandem immunoprecipitation (sequential IP) to improve specificity

  • Use biotin-labeled secondary antibodies with streptavidin-conjugated beads for signal enhancement

  • Consider proximity-dependent biotin identification (BioID) by fusing CYP83A1 with a biotin ligase

• Sample concentration methods:

  • Pool biological replicates when appropriate

  • Use tissues with highest CYP83A1 expression (based on promoter-GUS studies)

  • Consider enriching for appropriate subcellular fractions (microsomal fractions for ER-associated proteins)

• Validation approaches:

  • Include spike-in controls with recombinant CYP83A1 protein

  • Use cyp83a1 knockout mutants as negative controls

  • Perform parallel IP with antibodies against known CYP83A1-interacting proteins

How can CYP83A1 antibodies contribute to understanding plant defense mechanisms against powdery mildew?

CYP83A1 antibodies offer promising tools for advancing understanding of plant defense mechanisms against powdery mildew, particularly given the enhanced resistance observed in cyp83a1 mutants :

• Defense-related protein complex identification: Using CYP83A1 antibodies for co-immunoprecipitation followed by mass spectrometry can reveal protein interaction networks that connect glucosinolate metabolism to defense signaling.

• Spatio-temporal profiling during infection: Immunohistochemistry with CYP83A1 antibodies at different time points and locations during powdery mildew infection can map dynamic changes in protein distribution.

• Comparative proteomics approach: Quantitative Western blotting using CYP83A1 antibodies can establish protein abundance differences between susceptible wild-type plants and resistant cyp83a1 mutants before and during infection.

• Regulatory network mapping: ChIP-seq using antibodies against transcription factors predicted to regulate CYP83A1 can identify changes in transcriptional regulation during pathogen challenge.

• Protein-metabolite relationship analysis: Combining CYP83A1 immunoprecipitation with metabolite profiling can establish connections between enzyme levels and accumulation of defense compounds like camalexin, which accumulates at higher levels in cyp83a1-3 mutants .

Research has established that the enhanced resistance of cyp83a1-3 mutants requires NPR1, EDS1, and PAD4, but not SID2 or EDS5, placing CYP83A1 in a specific defense signaling context that can be further elucidated using antibody-based approaches .

What role can CYP83A1 antibodies play in investigating metabolic channeling in glucosinolate biosynthesis?

CYP83A1 antibodies provide sophisticated tools for investigating the hypothesis of metabolic channeling in glucosinolate biosynthesis:

• Multiprotein complex identification: Employ gentle co-immunoprecipitation with CYP83A1 antibodies combined with chemical crosslinking to capture transient protein interactions that may form metabolic channels.

• Proximity labeling approaches: Combine CYP83A1 antibodies with techniques like BioID or APEX2 proximity labeling to identify proteins in close physical proximity that may participate in substrate channeling.

• Superresolution microscopy with immunolabeling: Use techniques like STORM or PALM with CYP83A1 antibodies to visualize potential enzyme clusters at nanometer resolution, potentially revealing organized enzyme complexes.

• In situ enzyme activity correlations: Combine immunolocalization of CYP83A1 with activity-based protein profiling to determine if localized enzyme concentrations correlate with pathway activity.

• Protein-membrane interaction analysis: Use CYP83A1 antibodies in conjunction with membrane fractionation to investigate if metabolic channeling occurs through organization of pathway enzymes in specific membrane microdomains.

• Dynamic interaction studies: Implement Förster resonance energy transfer (FRET) between fluorescently labeled antibodies against CYP83A1 and other pathway enzymes to detect close associations in living cells.

Evidence for channeling comes from studies showing that both CYP83A1 and CYP83B1 metabolize oximes to thiohydroximates through transient S-alkylthiohydroximate intermediates, which are highly reactive and might require protected transfer to downstream enzymes .

How might antibody engineering enhance CYP83A1 research applications?

Advanced antibody engineering approaches can significantly enhance CYP83A1 research applications:

• Epitope-specific monoclonal antibody development: Generate antibodies targeting unique regions of CYP83A1 not present in CYP83B1 to eliminate cross-reactivity issues that complicate interpretation of results given their 63% sequence identity .

• Conformation-specific antibodies: Develop antibodies that selectively recognize active vs. inactive conformations of CYP83A1, enabling researchers to assess the functional state of the enzyme in different conditions.

• Intrabodies for in vivo studies: Engineer antibody fragments (scFv or nanobodies) that function intracellularly to track or modulate CYP83A1 activity in living plant cells without disruption.

• Bispecific antibodies: Create antibodies that simultaneously bind CYP83A1 and another glucosinolate biosynthetic enzyme to specifically detect enzyme complexes and study metabolic channeling.

• Antibody-enzyme fusions: Develop fusion proteins combining CYP83A1 antibody fragments with enzymes like HRP or alkaline phosphatase for direct detection without secondary antibodies.

• Site-specific conjugation: Implement site-specific labeling of antibodies with fluorophores or quantum dots at defined positions to maximize antigen binding while providing optimal signal for advanced microscopy techniques.

• Antibody immobilization strategies: Develop oriented antibody immobilization methods to maximize antigen capture efficiency in immunoprecipitation and chromatin immunoprecipitation experiments.

Implementation of these advanced antibody engineering approaches would provide researchers with more precise tools to investigate the complex interplay between glucosinolate biosynthesis, auxin homeostasis, and plant defense mechanisms mediated by CYP83A1 .

What are the optimal conditions for heterologous expression of CYP83A1 for antibody validation?

Optimizing heterologous expression of CYP83A1 for antibody validation requires careful consideration of several factors:

• Expression system selection:

  • Yeast (Saccharomyces cerevisiae) systems have been successfully used for functional CYP83A1 expression

  • Consider using WAT11 or other yeast strains engineered to express NADPH-cytochrome P450 reductase

  • E. coli systems with specialized vectors (pCWori+) containing N-terminal modifications can be used for producing larger quantities of protein for antibody production

  • Insect cell expression (Sf9, High Five) may improve protein folding and yield functional enzyme

• Construct optimization:

  • Include a C-terminal affinity tag (His6 or FLAG) for purification while ensuring it doesn't interfere with antibody epitopes

  • Consider N-terminal modifications to improve expression (removal of membrane-binding domain or fusion with soluble partners)

  • Codon optimization for the chosen expression system

  • Include TEV protease cleavage sites to remove tags after purification if necessary

• Expression conditions:

  • For yeast: induce with galactose at 30°C for 12-24 hours

  • Supplement media with δ-aminolevulinic acid (0.5 mM) as heme precursor

  • Add 5% glycerol to stabilize protein during expression

  • Consider lower temperatures (16-20°C) during induction phase to improve folding

• Purification strategy:

  • Use detergent mixtures (0.1% Triton X-100, 0.1% sodium cholate) for effective solubilization

  • Implement two-step purification (affinity chromatography followed by gel filtration)

  • Include glycerol (10%) and reducing agents in all buffers

  • Avoid freeze-thaw cycles that might denature the protein

• Functional verification:

  • Confirm enzyme activity using the aliphatic oxime conversion assay

  • Verify heme incorporation by measuring the CO-difference spectrum

  • Test substrate binding using spectral shift assays

This optimized expression and purification approach will provide high-quality CYP83A1 protein for antibody validation, ensuring that the antibodies recognize native conformations of the enzyme.

How can researchers distinguish between CYP83A1 and CYP83B1 in immunological studies?

Distinguishing between the closely related enzymes CYP83A1 and CYP83B1 (63% sequence identity) in immunological studies requires strategic approaches :

• Epitope mapping and antibody selection:

  • Perform sequence alignment between CYP83A1 and CYP83B1 to identify unique regions

  • Generate peptide-specific antibodies against non-conserved regions

  • Develop monoclonal antibodies with defined epitope specificity

  • Use epitope tagging in genetic complementation constructs when studying native plant systems

• Validation using genetic resources:

  • Test antibodies against protein extracts from cyp83a1 and cyp83b1 single knockout mutants

  • Use double knockouts (cyp83a1/cyp83b1) as negative controls

  • Employ transgenic lines with epitope-tagged versions of each protein

  • Test against heterologously expressed recombinant proteins of both enzymes

• Cross-adsorption techniques:

  • Pre-incubate antibodies with recombinant protein of the non-target enzyme to remove cross-reactive antibodies

  • Perform sequential immunoprecipitation to deplete cross-reactive species

  • Use competitive ELISA to quantify cross-reactivity

• Differential detection strategies:

  • Exploit differences in molecular weight (if present) using high-resolution SDS-PAGE

  • Use 2D gel electrophoresis to separate based on both pI and molecular weight

  • Implement multiplexed detection with differently labeled antibodies against unique epitopes

  • Combine with mass spectrometry for definitive identification

• Expression pattern differentiation:

  • Take advantage of differential tissue expression patterns

  • Utilize the different promoter responses (CYP83B1 has auxin-responsive elements while CYP83A1 does not)

  • Analyze samples from conditions known to differentially regulate these enzymes

By implementing these approaches, researchers can achieve specific detection of CYP83A1 despite its high similarity to CYP83B1, enabling accurate characterization of its unique roles in glucosinolate biosynthesis and plant defense.

What methodological considerations are important when studying CYP83A1 post-translational modifications using antibodies?

Investigating post-translational modifications (PTMs) of CYP83A1 using antibodies requires specialized methodological considerations:

• Sample preparation optimization:

  • Rapidly harvest and flash-freeze tissue to preserve labile modifications

  • Include specific inhibitors in extraction buffers (phosphatase inhibitors for phosphorylation, deubiquitinase inhibitors for ubiquitination)

  • Use mild detergents (0.5% digitonin) to maintain protein-protein interactions that might protect PTMs

  • Avoid reducing agents when studying disulfide bonds or redox-sensitive modifications

• PTM-specific antibody selection:

  • Use commercial phospho-specific antibodies for common motifs in combination with CYP83A1 immunoprecipitation

  • Consider generating custom antibodies against predicted PTM sites in CYP83A1

  • Use antibodies specific for ubiquitin, SUMO, or other modifiers in conjunction with CYP83A1 antibodies

  • Validate PTM-specific antibodies using appropriate controls (phosphatase-treated samples, mutagenesis of modification sites)

• Enrichment strategies:

  • Implement two-step immunoprecipitation: first with CYP83A1 antibodies, then with PTM-specific antibodies

  • Use phospho-peptide enrichment (TiO2, IMAC) after CYP83A1 immunoprecipitation and digestion

  • Apply ubiquitin remnant profiling for identifying ubiquitination sites

  • Consider click chemistry approaches for studying glycosylation or lipid modifications

• Detection methodologies:

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Implement multiplexed Western blotting with different fluorophores for total vs. modified protein

  • Apply Native PAGE to preserve intact protein complexes that might depend on PTMs

  • Consider hydrogen-deuterium exchange mass spectrometry after immunoprecipitation to identify structural changes induced by PTMs

• Functional correlation approaches:

  • Combine PTM detection with enzyme activity assays to correlate modifications with functional changes

  • Use site-directed mutagenesis to create non-modifiable variants (e.g., S→A for phosphorylation sites)

  • Apply temporal analysis during stress responses or developmental transitions to identify regulatory PTMs