CYP90B1 Antibody

What is CYP90B1 and why is it important in plant research?

CYP90B1 (also known as DWF4) is a cytochrome P450 monooxygenase that catalyzes the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the brassinosteroid (BR) biosynthetic pathway in plants . This enzyme is particularly significant because it:

• Preferentially catalyzes the conversion of campesterol (CR) to 22-hydroxycampesterol with remarkably high efficiency (325 times greater than for campestanol)

• Exhibits substrate specificity for sterols with a double bond at positions C-5 and C-6

• Utilizes molecular oxygen to insert one oxygen atom into the substrate while reducing the second into a water molecule, with electrons provided by NADPH via cytochrome P450 reductase

• Functions as a rate-limiting step in BR biosynthesis, making it a key regulatory point for plant growth and development

When studying plant growth regulation, brassinosteroid signaling, or hormone biosynthesis pathways, CYP90B1 represents a critical target for investigation.

What technical applications are CYP90B1 antibodies suitable for?

Based on the applications of comparable cytochrome P450 antibodies, CYP90B1 antibodies would typically be suitable for:

• Western blotting (WB): For quantitative analysis of protein expression levels

• Immunocytochemistry/Immunofluorescence (ICC/IF): For subcellular localization studies in fixed cells

• Immunohistochemistry (IHC): For tissue-specific expression analysis in plant sections

• Co-immunoprecipitation (Co-IP): For protein interaction studies

When selecting applications, researchers should verify the validation data for their specific antibody. As with other cytochrome P450 enzymes, proper microsomal preparation techniques are often required for successful detection of membrane-bound CYP90B1.

How should I optimize Western blot conditions for CYP90B1 detection?

For optimal Western blot detection of CYP90B1:

• Sample preparation:

  • Prepare microsomes from plant tissues since CYP90B1 is a membrane-associated protein

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

  • Avoid boiling samples (heat at 37°C for 30 minutes instead) to prevent aggregation of membrane proteins

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membranes (rather than nitrocellulose) for better retention of hydrophobic proteins

  • Use a wet transfer system at low voltage (30V) overnight at 4°C for efficient transfer of membrane proteins

• Antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature

  • Dilute primary antibody according to manufacturer recommendations (typically 1/500 to 1/2000)

  • Incubate with primary antibody overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody at 1/5000 to 1/10000 dilution

• Expected results:

  • The predicted molecular weight for CYP90B1 is approximately 56 kDa (similar to other P450 enzymes)

  • Consider using positive controls such as microsomes from plants overexpressing CYP90B1

How do I validate the specificity of a CYP90B1 antibody?

To validate CYP90B1 antibody specificity:

• Positive and negative controls:

  • Use wild-type plant tissue and cyp90b1 (dwf4) mutant tissues in parallel

  • Include recombinant CYP90B1 protein as a positive control if available

  • Test expression in heterologous systems (e.g., E. coli or insect cells expressing CYP90B1)

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application

  • A significant reduction in signal indicates specificity for the target epitope

• Molecular validation:

  • Compare protein expression with transcript levels via RT-PCR

  • Verify detection in plants with known CYP90B1 induction (e.g., BR-deficient mutants or plants treated with BR biosynthesis inhibitors)

• Cross-reactivity assessment:

  • Test the antibody against closely related CYP90 family proteins (CYP90C1, CYP90D1)

  • Evaluate detection in multiple plant species based on sequence conservation

How can I design experiments to correlate CYP90B1 protein levels with brassinosteroid biosynthesis activity?

To establish relationships between CYP90B1 protein abundance and its enzymatic activity:

• Combined protein and metabolite analysis:

  • Quantify CYP90B1 protein levels by Western blot using calibrated standards

  • Simultaneously measure endogenous brassinosteroid levels using GC-MS or LC-MS/MS

  • Analyze both parameters across developmental stages or in response to environmental stimuli

• Enzymatic activity correlation:

  • Prepare microsomes from plant tissues and quantify CYP90B1 protein

  • Perform in vitro CYP90B1 activity assays using campesterol as substrate

  • Measure the formation of 22-hydroxycampesterol using appropriate analytical methods

  • Calculate specific enzyme activity (activity per unit of protein)

• Experimental manipulations:

  • Compare wild-type plants with CYP90B1 overexpression lines and partial knockdown lines

  • Treat plants with BR biosynthesis inhibitors (uniconazole or brassinazole) that target CYP90B1

  • Analyze recovery of both protein levels and enzyme activity after inhibitor removal

• Data presentation:

  • Create correlation plots between protein abundance and enzymatic activity

  • Present data in a table format similar to:

What approaches can be used to study post-translational modifications of CYP90B1?

Post-translational modifications (PTMs) of CYP90B1 can be investigated using:

• Phosphorylation analysis:

  • Immunoprecipitate CYP90B1 using validated antibodies

  • Analyze samples by:
    a) Western blot with phospho-specific antibodies
    b) Phosphoprotein staining (e.g., Pro-Q Diamond)
    c) Mass spectrometry analysis of phosphopeptides

  • Compare samples from plants under different conditions (e.g., light/dark, stressed/unstressed)

• Ubiquitination and protein stability:

  • Treat plants with proteasome inhibitors (MG132)

  • Immunoprecipitate CYP90B1 and probe with anti-ubiquitin antibodies

  • Perform cycloheximide chase assays to measure protein half-life

  • Compare protein degradation kinetics in different genetic backgrounds or conditions

• Glycosylation assessment:

  • Treat immunoprecipitated CYP90B1 with glycosidases

  • Observe mobility shifts by Western blot

  • Use lectin blotting to detect specific glycan structures

• PTM site identification and mutation:

  • Identify modification sites by mass spectrometry

  • Generate site-directed mutants (e.g., S→A for phosphorylation sites)

  • Express mutant proteins in plants and assess:
    a) Protein stability and localization
    b) Enzymatic activity in microsomal assays
    c) Ability to complement cyp90b1/dwf4 mutant phenotypes

How can CYP90B1 protein-protein interactions be characterized in plant systems?

To investigate CYP90B1 interactions with other proteins:

• Co-immunoprecipitation approaches:

  • Use anti-CYP90B1 antibodies to pull down native protein complexes

  • Validate interactions by reciprocal co-IP with antibodies against suspected partners

  • Analyze isolated complexes by mass spectrometry to identify novel interactors

  • Focus particularly on interactions with NADPH-cytochrome P450 reductase, which is required for electron transfer

• Bimolecular fluorescence complementation (BiFC):

  • Create fusion constructs of CYP90B1 and potential interactors with split YFP fragments

  • Express in plant protoplasts or through transient expression

  • Visualize interactions through restored fluorescence

  • Include appropriate controls (e.g., known non-interactors)

• Förster resonance energy transfer (FRET):

  • Generate CYP90B1 fusions with donor fluorophores (e.g., CFP)

  • Create partner protein fusions with acceptor fluorophores (e.g., YFP)

  • Measure energy transfer as evidence of protein proximity

  • Calculate FRET efficiency and distance parameters

• Split-ubiquitin yeast two-hybrid for membrane proteins:

  • Use this specialized Y2H system designed for membrane proteins like CYP90B1

  • Screen libraries to identify novel interaction partners

  • Validate interactions in planta using the methods described above

• Proximity-dependent biotin identification (BioID):

  • Create CYP90B1-BioID fusion proteins

  • Express in plant systems and provide biotin

  • Identify biotinylated proximity partners by streptavidin pull-down and mass spectrometry

What methodologies can be employed to study the membrane localization and trafficking of CYP90B1?

To investigate CYP90B1 subcellular localization and membrane integration:

• Subcellular fractionation:

  • Isolate microsomal fractions from plant tissues

  • Further separate endoplasmic reticulum (ER) from other organelles using sucrose gradient centrifugation

  • Detect CYP90B1 by Western blot in different fractions

  • Use organelle-specific markers (e.g., BiP for ER, cytochrome c oxidase for mitochondria) as controls

• Fluorescence microscopy:

  • Generate CYP90B1-GFP fusion constructs

  • Express in plant cells through stable transformation or transient expression

  • Co-localize with organelle markers (e.g., ER-Tracker, MitoTracker)

  • Perform live cell imaging to track protein movement

• Immunogold electron microscopy:

  • Use CYP90B1 antibodies with gold-conjugated secondary antibodies

  • Visualize precise localization at ultrastructural level

  • Quantify gold particle distribution across cellular compartments

• Topology analysis:

  • Perform protease protection assays on microsomes

  • Use selective membrane permeabilization followed by immunodetection

  • Create epitope-tagged versions of CYP90B1 with tags in different domains

  • Determine which portions are accessible to antibodies in intact versus permeabilized membranes

• Monitoring trafficking:

  • Use photoactivatable or photoconvertible fluorescent protein fusions

  • Track movement after photoactivation in specific compartments

  • Employ inhibitors of vesicular trafficking to determine transport mechanisms

How do I resolve issues with low or inconsistent signal when detecting CYP90B1?

When facing difficulties detecting CYP90B1:

• Sample preparation optimization:

  • Ensure complete tissue disruption using appropriate homogenization methods for plant tissues

  • Add protease inhibitors immediately after tissue homogenization

  • Optimize microsome preparation protocol (consider differential centrifugation conditions)

  • Avoid freeze-thaw cycles of prepared samples

• Antibody-related solutions:

  • Test different antibody concentrations (perform a titration series)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try alternative detection systems (e.g., more sensitive ECL reagents, fluorescent secondary antibodies)

  • Consider using antibodies raised against different epitopes of CYP90B1

• Signal enhancement strategies:

  • Increase protein loading (while ensuring the gel runs properly)

  • Reduce washing stringency (shorter washes, lower detergent concentration)

  • Use signal enhancers compatible with your detection system

  • Consider enhanced chemiluminescence substrates with extended signal duration

• Expression level considerations:

  • CYP90B1 may be expressed at different levels across tissues and developmental stages

  • Test samples from tissues with known higher expression (e.g., actively growing tissues)

  • Use plants treated with factors known to upregulate CYP90B1 (e.g., specific transcription factors)

How can I distinguish between CYP90B1 and other related cytochrome P450 enzymes in my analyses?

To ensure specificity when analyzing CYP90B1:

• Antibody selection:

  • Choose antibodies raised against unique regions of CYP90B1 not conserved in related P450s

  • Verify antibody specificity using recombinant proteins or knockout mutants

  • Consider using epitope-tagged CYP90B1 constructs for unambiguous detection

• Experimental controls:

  • Include samples from cyp90b1/dwf4 mutant plants as negative controls

  • Use recombinant CYP90B1 protein as a positive control and size reference

  • Include other purified CYP90 family members to assess cross-reactivity

• Validation approaches:

  • Complement Western blot data with RT-PCR using gene-specific primers

  • Perform mass spectrometry analysis of immunoprecipitated proteins

  • Use multiple antibodies targeting different epitopes of CYP90B1

• Differential analysis:

  • Compare protein expression patterns with known tissue-specific expression data

  • Analyze responses to treatments that specifically induce CYP90B1 but not related enzymes

  • Examine enzymatic activity using substrate specificity differences (e.g., CYP90B1 has 325× higher activity with campesterol than campestanol)

How do genetic modifications in CYP90B1 affect antibody recognition and experimental design?

When working with modified CYP90B1 variants:

• Point mutations and antibody binding:

  • Mutations near antibody epitopes may reduce detection efficiency

  • If using antibodies against specific domains, verify that these regions remain intact in your mutant

  • For critical mutations, consider using multiple antibodies targeting different regions

• Truncated proteins:

  • Confirm that the epitope recognized by your antibody is present in truncated forms

  • Use antibodies raised against different regions to detect specific fragments

  • Consider size differences when interpreting Western blot results

• Tagged proteins:

  • Verify that tags do not interfere with antibody epitopes

  • Use tag-specific antibodies as alternative detection methods

  • Consider the impact of tags on protein folding, localization, and function

• Experimental design considerations:

  • Include appropriate negative controls (null mutants) and positive controls (wild-type)

  • When possible, use multiple detection methods to confirm results

  • For complementation studies, consider both protein level and functional recovery

• Recommendations for specific scenarios:

  • For T-DNA insertion lines: Map the insertion site relative to antibody epitopes

  • For point mutations: Test antibody reactivity with recombinant mutant proteins beforehand

  • For overexpression lines: Verify that antibody detection remains in the linear range

How can CYP90B1 antibodies be used to investigate cross-talk between brassinosteroid biosynthesis and other signaling pathways?

To study pathway integration using CYP90B1 antibodies:

• Hormone crosstalk analysis:

  • Treat plants with different hormones (auxin, gibberellin, ethylene, etc.)

  • Monitor CYP90B1 protein levels via Western blot

  • Compare protein changes with transcriptional responses

  • Perform these experiments in hormone signaling mutants to define pathway dependencies

• Stress response integration:

  • Subject plants to various stresses (drought, salt, temperature)

  • Analyze CYP90B1 protein abundance in different tissues

  • Correlate with BR biosynthesis activity and physiological responses

  • Use immunolocalization to determine if stress alters subcellular distribution

• Light signaling connections:

  • Given the known relationship between CYP90B1/DWF4 and light-responsive transcription factors (like PIFs)

  • Compare CYP90B1 levels under different light conditions (quality, intensity, duration)

  • Analyze protein expression in photoreceptor mutants

  • Use ChIP assays with transcription factor antibodies to study direct regulation of CYP90B1

• Developmental regulation:

  • Immunolocalize CYP90B1 across developmental stages and tissues

  • Compare with expression patterns of key developmental regulators

  • Use co-immunoprecipitation to identify stage-specific interacting partners

What techniques can be used to study the post-translational regulation of CYP90B1 enzymatic activity?

To investigate regulation beyond transcriptional control:

• In vitro activity modulation:

  • Immunopurify CYP90B1 from plants under different conditions

  • Perform enzymatic assays with campesterol substrate

  • Compare specific activity (activity normalized to protein amount)

  • Test effects of potential regulators added to the reaction mixture

• Protein modification analysis:

  • Use phospho-specific antibodies to detect phosphorylated CYP90B1

  • Treat samples with phosphatases to assess impact on activity

  • Perform mass spectrometry to identify modification sites

  • Create site-directed mutants to confirm functional significance

• Protein-protein interaction effects:

  • Identify CYP90B1 interactors through co-immunoprecipitation

  • Reconstitute activity assays with and without interacting proteins

  • Use proximity labeling approaches (BioID) to identify transient interactors

  • Test if POR (P450 reductase) association is regulated under different conditions

• Membrane environment influences:

  • Investigate if localization within specialized ER domains affects activity

  • Test effects of membrane lipid composition on enzyme function

  • Analyze potential associations with lipid rafts or detergent-resistant membranes

  • Consider sterol-dependent regulation mechanisms

How can I design experiments to study the impact of CYP90B1 inhibitors using antibody-based approaches?

When investigating CYP90B1 inhibitors like brassinazole and uniconazole :

• Inhibitor binding studies:

  • Use thermal shift assays with purified CYP90B1 to assess inhibitor binding

  • Perform competitive binding studies between substrates and inhibitors

  • Correlate structural insights from crystal structures with functional impacts

• Protein stability and turnover:

  • Treat plants with inhibitors and monitor CYP90B1 protein levels over time

  • Perform cycloheximide chase experiments to determine if inhibitors affect protein half-life

  • Investigate if inhibitor binding alters susceptibility to degradation pathways

• Subcellular localization effects:

  • Examine if inhibitor treatment changes CYP90B1 localization

  • Use immunofluorescence or CYP90B1-GFP fusions

  • Determine if inhibition triggers relocalization or aggregation

• Experimental design considerations:

  • Include dose-response analyses with both biochemical and physiological readouts

  • Compare time courses of inhibitor effects on protein activity vs. abundance

  • Design recovery experiments by inhibitor removal to distinguish temporary from permanent effects

How can CYP90B1 antibodies be used in conjunction with transcription factor analysis to study brassinosteroid biosynthesis regulation?

To investigate transcriptional regulation of CYP90B1:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Use antibodies against transcription factors like BES1, COG1, PIF4, or PIF5

  • Perform ChIP followed by qPCR targeting the CYP90B1/DWF4 promoter

  • Compare binding under different conditions (light/dark, hormone treatments)

  • Validate with reporter gene assays using CYP90B1 promoter constructs

• Correlation analyses:

  • Monitor transcription factor abundance by Western blot

  • Simultaneously assess CYP90B1 protein levels

  • Create correlation plots between transcription factor levels and CYP90B1 abundance

  • Test causality in transcription factor mutants or overexpression lines

• Multi-level regulation studies:

  • Compare CYP90B1 transcript levels (by RT-qPCR) with protein abundance (by Western blot)

  • Assess if post-transcriptional mechanisms are involved in specific conditions

  • Calculate protein-to-mRNA ratios across experimental treatments

• Factor-specific approaches:

  • For COG1 (known to regulate CYP90B1 via PIF4/PIF5) :

    • Compare CYP90B1 protein levels in wild-type, cog1 mutant, and COG1 overexpression lines

    • Assess if COG1's effects on CYP90B1 are direct or mediated through other factors

    • Use inducible systems to determine the kinetics of CYP90B1 upregulation following transcription factor activation