CYP72C1 Antibody

What is CYP72C1 and what are its primary functions in plants?

CYP72C1 is a cytochrome P450 enzyme primarily found in Arabidopsis thaliana. It plays a crucial role in brassinosteroid (BR) inactivation and regulation of BR homeostasis. Unlike typical cytochrome P450s, CYP72C1 lacks carbon 26 hydroxylase activity and may inactivate brassinosteroids by hydroxylating carbons at positions other than C-26. This protein works in association with CYP734A1 to modulate photomorphogenesis in plants .

The gene has several synonym names including CHIBI 2 (CHI2), DWARFISH WITH LOW FERTILITY (DLF), SHRINK 1 (SHK1), and SUPPRESSOR OF PHYB-4 PROTEIN 7 (SOB7) . Overexpression of CYP72C1 results in plants exhibiting a dwarf phenotype similar to brassinolide-resistant mutants, while loss-of-function mutations lead to plants with elongated hypocotyls .

How do researchers confirm the specificity of anti-CYP72C1 antibodies?

Confirming antibody specificity for CYP72C1 requires multiple validation approaches:

• Western blot analysis: Run protein samples from wild-type and CYP72C1 knockout/knockdown plants side-by-side to verify band presence/absence at the expected molecular weight (typically using methods similar to those described for other cytochrome P450 antibodies) .

• Immunoprecipitation followed by mass spectrometry: Precipitate proteins using the anti-CYP72C1 antibody, then confirm identity through peptide sequencing.

• Pre-absorption control: Pre-incubate the antibody with purified recombinant CYP72C1 protein before immunostaining to demonstrate signal reduction.

• Cross-reactivity testing: Test against closely related cytochrome P450 family members to ensure specificity.

• Immunohistochemistry comparison: Compare staining patterns with known CYP72C1 expression patterns from transcriptomic data.

The three-dimensional structure of CYP72C1's active site notably lacks several conserved amino acids typically required for substrate hydroxylation, making antibody epitope selection particularly important for specificity .

What experimental applications are anti-CYP72C1 antibodies most commonly used for?

Anti-CYP72C1 antibodies are primarily used in the following research applications:

The antibody has proven particularly valuable for studying CYP72C1's role in brassinosteroid metabolism and its interactions with other components of phytohormone signaling pathways .

What are the optimal conditions for Western blot analysis using anti-CYP72C1 antibodies?

For optimal Western blot results with anti-CYP72C1 antibodies, researchers should consider the following protocol:

• Sample preparation:

  • Extract plant proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

  • Include reducing agents (5-10 mM DTT) to ensure proper protein denaturation

  • Heat samples at 95°C for 5 minutes before loading

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of CYP72C1 (predicted MW approximately 55-60 kDa)

  • Include positive controls (recombinant CYP72C1) and negative controls (extracts from CYP72C1 knockout plants)

• Transfer and blocking:

  • Transfer proteins to PVDF membranes (preferable to nitrocellulose for plant P450s)

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

• Antibody incubation:

  • Primary antibody dilution: 1:1000-1:2000 in blocking buffer, incubate overnight at 4°C

  • Wash 3-5 times with TBST

  • Secondary antibody (HRP-conjugated anti-rabbit IgG): 1:5000-1:10000, incubate for 1 hour at room temperature

• Detection:

  • Use chemiluminescent substrate for visualization

  • Expected band should appear at approximately 55-60 kDa

Similar methodology has been effectively employed for other plant cytochrome P450 proteins and can be adapted for CYP72C1 .

How should researchers troubleshoot non-specific binding when using anti-CYP72C1 antibodies?

When encountering non-specific binding with anti-CYP72C1 antibodies, consider the following troubleshooting approaches:

• Increase blocking stringency:

  • Extend blocking time to 2 hours

  • Try alternative blocking reagents (BSA, commercial blockers optimized for plant samples)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Optimize antibody dilution:

  • Perform a dilution series (1:500, 1:1000, 1:2000, 1:5000) to determine optimal signal-to-noise ratio

  • Consider longer incubation at higher dilutions (e.g., 48 hours at 4°C with 1:5000 dilution)

• Pre-absorb antibody:

  • Incubate antibody with protein extract from CYP72C1 knockout plants before use

  • This removes antibodies binding to non-specific plant proteins

• Modify washing conditions:

  • Increase wash duration and number (5 washes × 10 minutes)

  • Add higher salt concentration (up to 500 mM NaCl) in wash buffer

  • Include 0.1% SDS in wash buffer for more stringent conditions

• Evaluate fixation methods (for immunohistochemistry):

  • Compare aldehyde-based versus alcohol-based fixatives

  • Optimize antigen retrieval methods for plant tissues

Similar approaches have been successful in optimizing specificity for other plant cytochrome P450 antibodies .

What considerations are important when selecting anti-CYP72C1 antibodies for different plant species?

When selecting anti-CYP72C1 antibodies for cross-species applications, researchers should consider:

• Sequence homology analysis:

  • Perform sequence alignment of CYP72C1 orthologs across target species

  • Focus on antibodies raised against conserved epitopes for cross-species applications

  • For polyclonal antibodies, evaluate whether the immunogen region is conserved

• Domain-specific antibodies:

  • Select antibodies targeting functional domains that are more likely to be conserved

  • Avoid antibodies targeting regions with high sequence divergence

• Validation requirements:

  • Always validate in each new species before proceeding with experiments

  • Start with Western blot to confirm reactivity and specificity

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

• Production methods:

  • Recombinant fragment-based antibodies (like those targeting amino acids 50-400) generally offer better specificity than those raised against synthetic peptides

  • For novel species, consider custom antibody production using conserved epitopes

• Control experiments:

  • Include extracts from model species (Arabidopsis) as positive controls

  • Use pre-immune serum controls to assess background

  • Consider using transgenic plants expressing tagged versions of the target protein

The amino acid sequence of CYP72C1 contains regions that lack conserved features typically found in other cytochrome P450s, which may affect antibody cross-reactivity .

How can researchers effectively use anti-CYP72C1 antibodies to study brassinosteroid metabolism?

To investigate brassinosteroid metabolism using anti-CYP72C1 antibodies, researchers can implement these advanced approaches:

• Co-immunoprecipitation studies:

  • Use anti-CYP72C1 antibodies to precipitate the protein complex

  • Analyze interacting partners through mass spectrometry

  • Compare protein interactions under different hormonal conditions

  • Include appropriate controls (IgG, pre-immune serum)

• Chromatin immunoprecipitation (ChIP) analysis:

  • For studying transcription factors that regulate CYP72C1 expression

  • Use formaldehyde crosslinking followed by immunoprecipitation with antibodies against candidate transcription factors

  • Perform qPCR on CYP72C1 promoter regions to quantify binding

• Subcellular fractionation combined with immunoblotting:

  • Separate endoplasmic reticulum, cytosol, and microsomal fractions

  • Probe fractions with anti-CYP72C1 antibodies to determine localization

  • Monitor changes in localization upon hormone treatment

• Immunohistochemistry during development:

  • Track CYP72C1 protein levels across developmental stages

  • Correlate with brassinosteroid-responsive phenotypes

  • Compare with expression patterns of CYP734A1, its functional partner

• In vitro enzyme assays:

  • Immunopurify CYP72C1 using the antibody

  • Test activity on different brassinosteroid substrates

  • Analyze hydroxylation products by LC-MS/MS

This approach leverages CYP72C1's established role in brassinosteroid inactivation and homeostasis regulation, as documented in the literature .

What methodologies can distinguish the functions of CYP72C1 from other cytochrome P450s involved in brassinosteroid metabolism?

Distinguishing CYP72C1 function from other BR-metabolizing P450s requires sophisticated approaches:

• Differential inhibition studies:

  • Apply specific P450 inhibitors with varying selectivity

  • Combine with immunodetection to correlate enzyme levels with metabolic outcomes

  • Monitor brassinosteroid levels by LC-MS/MS

• Protein complex analysis:

  • Use anti-CYP72C1 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare interacting partners with those of other P450s (particularly CYP734A1)

  • Identify unique vs. shared protein complexes

• Site-directed mutagenesis combined with antibody detection:

  • Generate variants of CYP72C1 with mutations in the atypical active site residues

  • Express in heterologous systems or transform into cyp72c1 mutants

  • Use antibodies to confirm expression and analyze functional consequences

• Advanced microscopy techniques:

  • Perform co-localization studies using anti-CYP72C1 antibodies with other fluorescently labeled P450s

  • Employ proximity ligation assays to detect protein-protein interactions in situ

  • Use super-resolution microscopy to visualize microdomains within the ER

• Conditional expression systems:

  • Generate inducible CYP72C1 lines and monitor immediate changes in the brassinosteroid profile

  • Use anti-CYP72C1 antibodies to confirm expression timing and levels

  • Compare with similar systems for other P450s

CYP72C1 lacks several conserved amino acids typically present in substrate hydroxylation sites of cytochrome P450s, suggesting a unique mechanism of action compared to other BR-metabolizing enzymes .

What are the best practices for detecting post-translational modifications of CYP72C1 using specific antibodies?

Detecting post-translational modifications (PTMs) of CYP72C1 requires specialized approaches:

• PTM-specific antibody selection:

  • Use antibodies specifically targeting phosphorylated, ubiquitinated, or glycosylated forms

  • Combine with general anti-CYP72C1 antibodies in parallel experiments

• Two-dimensional gel electrophoresis workflow:

  • Separate proteins by isoelectric point followed by molecular weight

  • Perform Western blot with anti-CYP72C1 antibodies

  • Identify charge variants indicating phosphorylation or other modifications

  • Compare patterns under different physiological conditions

• Sequential immunoprecipitation approach:

  • First immunoprecipitation: use general anti-CYP72C1 antibody

  • Second immunoprecipitation: use PTM-specific antibodies (anti-phospho, anti-ubiquitin)

  • Alternatively, first precipitate with PTM antibodies, then probe with CYP72C1 antibodies

• Mass spectrometry validation:

  • Immunoprecipitate CYP72C1 using specific antibodies

  • Perform tryptic digestion and analyze peptides by LC-MS/MS

  • Map modifications to specific residues

  • Compare modifications under different experimental conditions

• In vitro kinase/phosphatase assays:

  • Immunopurify CYP72C1 using specific antibodies

  • Subject to kinase or phosphatase treatments

  • Monitor mobility shifts by Western blot

This methodology leverages techniques similar to those used for studying other cytochrome P450 modifications, adapted specifically for the unique structural features of CYP72C1 .

How can researchers address epitope masking issues when working with membrane-associated CYP72C1?

Epitope masking is a common challenge when studying membrane-associated proteins like CYP72C1. Researchers can employ these strategies:

• Optimize membrane protein extraction:

  • Use mild detergents (0.5-1% digitonin, DDM, or CHAPS) for native conformation preservation

  • Compare different solubilization methods (detergent types and concentrations)

  • Test specialized membrane protein extraction kits

  • Include proper controls to ensure complete extraction

• Epitope exposure techniques:

  • For fixed tissues: implement antigen retrieval protocols (heat-induced or enzymatic)

  • For Western blots: test various denaturation conditions (temperature, SDS concentration)

  • For immunoprecipitation: evaluate different antibody binding buffers

• Alternative antibody approaches:

  • Use antibodies targeting multiple epitopes (N-terminal, C-terminal, internal domains)

  • Consider using antibodies against tags in recombinant systems (His, FLAG, HA)

  • Develop conformational antibodies that recognize native structures

• Crosslinking experiments:

  • Employ membrane-permeable crosslinkers to fix protein interactions before extraction

  • Use proximity labeling methods (BioID, APEX) combined with antibody detection

• Domain-specific accessibility analysis:

  • Generate constructs with tags in different protein domains

  • Compare detection efficiency using tag-specific vs. CYP72C1-specific antibodies

  • Map accessible epitopes under different conditions

Similar approaches have been effective for studying other membrane-bound cytochrome P450 enzymes in various experimental systems .

What emerging technologies are enhancing the specificity and application range of anti-CYP72C1 antibodies?

Several cutting-edge technologies are expanding the capabilities of anti-CYP72C1 antibody research:

• Single-domain antibodies (nanobodies):

  • Development of camelid-derived nanobodies against CYP72C1

  • Advantages: smaller size, better access to concealed epitopes, improved stability

  • Applications: super-resolution microscopy, in vivo imaging

• Recombinant antibody engineering:

  • Custom-designed antibody fragments with enhanced specificity

  • Site-directed mutagenesis to improve affinity and reduce cross-reactivity

  • Humanized antibodies for longer-term experimental applications

• Antibody-enzyme fusion proteins:

  • Direct conjugation of reporter enzymes to anti-CYP72C1 antibodies

  • Applications: enhanced detection sensitivity, simplified protocols

  • Examples: HRP-conjugated, alkaline phosphatase-conjugated antibodies

• Multiplexed antibody arrays:

  • Simultaneous detection of CYP72C1 along with other brassinosteroid pathway components

  • Quantitative analysis of multiple proteins from limited samples

  • Integration with microfluidic delivery systems

• In situ proximity labeling:

  • Antibody-guided enzyme proximity labeling (APEX, BioID)

  • Applications: mapping protein interaction networks in native environments

  • Advantage: captures transient interactions difficult to detect by traditional methods

The development of these technologies builds on methods similar to those used for other cytochrome P450 research, with specific adaptations for plant systems and the unique properties of CYP72C1 .

How can structural biology approaches inform better anti-CYP72C1 antibody design and application?

Structural biology can significantly enhance anti-CYP72C1 antibody design and application through:

• Epitope mapping and optimization:

  • Use structural data to identify surface-exposed regions unique to CYP72C1

  • Design antibodies targeting regions with minimal homology to other P450s

  • Focus on regions stable across different functional states

• Conformation-specific antibody development:

  • Generate antibodies recognizing specific functional states (substrate-bound, active, inactive)

  • Use structural data to select conformational epitopes

  • Applications: monitoring enzyme activity states in vivo

• Rational antibody engineering:

  • Model antibody-antigen interaction using structural data

  • Optimize complementarity-determining regions for improved affinity

  • Design antibodies with minimal cross-reactivity to related P450s

• Structure-guided functional analysis:

  • Use structural knowledge of the atypical active site to interpret antibody-based functional studies

  • Design experiments targeting specific structural features unique to CYP72C1

  • Compare with structural characteristics of other BR-metabolizing P450s

• Antibody-facilitated crystallography:

  • Use antibody fragments to stabilize CYP72C1 for crystallization

  • Apply antibody-mediated purification to obtain homogeneous protein samples

  • Leverage antibody-antigen co-crystals for structural determination

CYP72C1's atypical active site, which lacks several conserved amino acids typically required for substrate hydroxylation, makes structural approaches particularly valuable for understanding its unique mechanism and designing specific research tools .

How can CYP72C1 antibodies contribute to comparative studies across plant hormone signaling pathways?

Anti-CYP72C1 antibodies can facilitate comparative analysis of hormone signaling networks through:

• Multi-pathway protein complex analysis:

  • Immunoprecipitate CYP72C1 complexes under different hormonal treatments

  • Identify components shared between brassinosteroid and other hormone pathways

  • Map signaling crosstalk points at the protein level

• Co-expression correlation studies:

  • Use immunohistochemistry with anti-CYP72C1 antibodies alongside markers for other hormone pathways

  • Quantify co-localization patterns in different tissues and developmental stages

  • Correlate protein levels with hormone measurements

• Protein modification comparative analysis:

  • Track post-translational modifications of CYP72C1 in response to multiple hormones

  • Compare modification patterns with those of proteins in other hormone pathways

  • Identify common regulatory mechanisms

• Differential expression in hormone mutants:

  • Analyze CYP72C1 protein levels in mutants of various hormone pathways

  • Use quantitative Western blotting with anti-CYP72C1 antibodies

  • Create comprehensive protein expression maps across hormone signaling networks

• Hormone response protein interactome mapping:

  • Combine antibody-based pulldowns with proximity labeling approaches

  • Construct interaction networks under different hormonal conditions

  • Identify network hubs and regulatory nodes

This approach leverages CYP72C1's established role in brassinosteroid inactivation while exploring its connections to broader hormonal networks regulating plant development and stress responses .

What are the best strategies for using anti-CYP72C1 antibodies in studying plant responses to environmental stresses?

For studying environmental stress responses using anti-CYP72C1 antibodies, researchers should consider:

• Quantitative immunoblotting protocol:

  • Collect plant samples under defined stress conditions (drought, salt, temperature, pathogens)

  • Perform quantitative Western blotting using anti-CYP72C1 antibodies

  • Include appropriate loading controls and standard curves

  • Correlate protein levels with brassinosteroid measurements and phenotypic responses

• Tissue-specific expression analysis:

  • Use immunohistochemistry to map CYP72C1 protein distribution under stress

  • Compare with normal conditions to identify stress-responsive tissues

  • Correlate with known stress response markers

• Time-course experiments:

  • Track CYP72C1 protein levels across stress exposure timeline

  • Use consistent sampling and extraction protocols

  • Correlate with transcriptional changes and metabolite profiles

• Stress-induced protein interaction studies:

  • Perform co-immunoprecipitation with anti-CYP72C1 antibodies under stress conditions

  • Compare interacting partners between normal and stress conditions

  • Identify stress-specific protein complexes

• Transgenic approaches combined with antibody detection:

  • Generate plants with altered CYP72C1 expression (overexpression, RNAi)

  • Confirm expression levels using anti-CYP72C1 antibodies

  • Assess stress tolerance phenotypes and correlate with protein levels

This methodology builds on CYP72C1's role in brassinosteroid metabolism, which is known to influence plant stress responses, while providing protein-level insights not obtainable through transcriptomic approaches alone .

How can researchers effectively combine genetic and immunological approaches when studying CYP72C1 function?

Integrating genetic and immunological approaches provides powerful insights into CYP72C1 function:

• Complementation analysis with immunodetection:

  • Transform cyp72c1 mutants with modified CYP72C1 variants

  • Use antibodies to confirm and quantify protein expression

  • Correlate protein levels with phenotypic rescue

  • Analyze structure-function relationships with domain deletions/mutations

• CRISPR-engineered variants with epitope tags:

  • Create endogenous tagged versions of CYP72C1

  • Compare detection efficiency between anti-tag and anti-CYP72C1 antibodies

  • Validate modifications have minimal impact on function

  • Use for in vivo tracking of protein dynamics

• Quantitative trait locus (QTL) analysis with protein quantification:

  • Identify natural CYP72C1 variants through genetic mapping

  • Use antibodies to quantify protein levels across accessions

  • Correlate protein abundance with phenotypic variation

  • Identify post-transcriptional regulatory mechanisms

• Conditional genetic systems with antibody monitoring:

  • Develop inducible CYP72C1 expression/suppression systems

  • Use antibodies to confirm timing and extent of expression changes

  • Track immediate downstream effects on protein networks

  • Monitor protein stability under different conditions

• Multi-omics integration:

  • Combine antibody-based proteomics with transcriptomics and metabolomics

  • Create comprehensive maps of CYP72C1 regulation and function

  • Identify discrepancies between transcript and protein levels

  • Discover post-transcriptional regulatory mechanisms

This integrated approach leverages both the specificity of genetic manipulation and the direct protein-level insights provided by immunological methods to build a comprehensive understanding of CYP72C1 function in plant development and brassinosteroid metabolism .