CYP71B35 Antibody

What types of CYP71B35 antibodies are available and what are their specifications?

CYP71B35 antibodies are available as polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana CYP71B35 protein. These antibodies are typically suitable for ELISA and Western blot applications and react specifically with Arabidopsis thaliana samples . Most commercially available options are provided in liquid form, stored in buffers containing glycerol (approximately 50%), PBS (0.01M, pH 7.4), and preservatives such as Proclin 300 (0.03%) . These antibodies are generally purified using antigen affinity methods to enhance specificity.

When selecting a CYP71B35 antibody, researchers should consider:

• Clonality (polyclonal vs monoclonal)

• Host species (commonly rabbit)

• Validated applications (ELISA, WB, IHC, ICC)

• Storage requirements (-20°C or -80°C, avoiding repeated freeze-thaw cycles)

• Lead time for acquisition (typically 14-16 weeks for made-to-order antibodies)

How should I validate the specificity of a CYP71B35 antibody?

Proper validation of CYP71B35 antibodies is crucial for ensuring experimental reliability. Based on established validation protocols for antibodies, researchers should:

• Perform Western blot analysis to confirm a single band of appropriate molecular weight

• Include negative controls using tissue/cells from CYP71B35 knockout or knockdown plants

• Test the antibody in tissues known not to express CYP71B35

• Compare reactivity with recombinant CYP71B35 protein as a positive control

• Perform immunoprecipitation followed by mass spectrometry to confirm target identity

A comprehensive validation approach should include multiple complementary techniques. For example, in a study evaluating antibody specificity against NF-κB-subunit p65, researchers tested six different antibodies using both immunocytochemistry and Western blots, revealing significant differences in specificity between antibodies targeting the same protein .

What are the essential controls for experiments using CYP71B35 antibodies?

When designing experiments with CYP71B35 antibodies, proper controls are essential to demonstrate specificity and rule out false positive results. Based on flow cytometry experimental design principles, which can be applied to other antibody-based techniques, researchers should include:

• Unstained controls: To establish baseline autofluorescence levels from plant tissues

• Negative controls: Samples known not to express CYP71B35

• Isotype controls: Antibodies of the same class as the primary antibody but with no known specificity for CYP71B35

• Secondary antibody controls: Samples treated with only labeled secondary antibody to assess non-specific binding

Additionally, appropriate blocking steps are crucial to minimize background and improve signal-to-noise ratio. For plant samples, researchers should use blocking buffer containing 10% normal serum from the same host species as the labeled secondary antibody. Importantly, this serum should NOT be from the same host species as the primary antibody to avoid non-specific signals .

What are the common applications for CYP71B35 antibodies in plant research?

CYP71B35 antibodies can be employed in various research applications to study the expression, localization, and function of this cytochrome P450 enzyme:

• Western Blotting (WB): For quantitative analysis of CYP71B35 expression across different tissues or experimental conditions

• Enzyme-Linked Immunosorbent Assay (ELISA): For high-throughput quantification of CYP71B35 levels

• Immunohistochemistry (IHC): To visualize tissue distribution patterns

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

• Immunoprecipitation (IP): To isolate CYP71B35 and identify potential interaction partners

When selecting methods, researchers should consider that cytochrome P450 enzymes like CYP71B35 are typically membrane-associated proteins, which may influence extraction conditions and detection methods.

What are the optimal storage and handling conditions for CYP71B35 antibodies?

To maintain antibody functionality and prevent degradation:

• Store antibodies at recommended temperatures (-20°C or -80°C) in aliquots to avoid repeated freeze-thaw cycles

• Include cryoprotectants like glycerol (typically 50%) in storage buffer

• Add preservatives (e.g., 0.03% Proclin 300) to prevent microbial contamination

• Use appropriate buffers (e.g., 0.01M PBS, pH 7.4) to maintain protein stability

• When thawing, allow antibodies to reach room temperature gradually before use

• Avoid exposure to direct light, especially for conjugated antibodies

Following these practices can significantly extend the shelf-life and performance of CYP71B35 antibodies in research applications.

How can I optimize immunoprecipitation protocols with CYP71B35 antibodies?

Immunoprecipitation (IP) with CYP71B35 antibodies requires careful optimization to efficiently isolate this membrane-associated cytochrome P450 enzyme:

• Lysis buffer optimization: Use buffers containing mild detergents (0.5-1% NP-40, Triton X-100, or digitonin) to solubilize membrane proteins while preserving protein-protein interactions

• Cross-linking considerations: For transient interactions, consider using membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate]) prior to cell lysis

• Antibody coupling strategies: For cleaner results, consider covalently coupling CYP71B35 antibodies to protein A/G beads using dimethyl pimelimidate (DMP)

• Optimized wash conditions: Balance between stringency (to reduce non-specific binding) and gentleness (to maintain specific interactions)

• Elution methods: Consider competitive elution with antigenic peptides for native conditions or SDS elution for denaturing conditions

For plant samples specifically, cell wall disruption is a critical step. Using a combination of mechanical disruption and enzymatic digestion can improve protein extraction efficiency. Additionally, including protease inhibitors and performing all steps at 4°C can minimize protein degradation .

What approaches can overcome challenges in detecting low-abundance CYP71B35 protein in plant tissues?

Detecting low-abundance proteins like CYP71B35 in complex plant tissues presents several challenges:

• Sample enrichment strategies:

  • Perform subcellular fractionation to isolate microsomal fractions where cytochrome P450 enzymes typically reside

  • Use differential centrifugation to separate membrane fractions

  • Consider affinity purification to concentrate CYP71B35 prior to detection

• Signal amplification methods:

  • Employ high-sensitivity chemiluminescent substrates for Western blots

  • Use tyramide signal amplification (TSA) for immunohistochemistry

  • Consider biotin-streptavidin systems to enhance detection sensitivity

• Advanced detection systems:

  • Utilize single-cell encapsulation technologies for analyzing low-abundance proteins in specific cell types

  • Apply micro-scale antibody production and screening methods for increased sensitivity

  • Consider employing proximity-based detection methods like proximity ligation assay (PLA)

• Optimization of protein extraction:

  • Test different extraction buffers specific for membrane proteins

  • Include appropriate detergents and chaotropic agents to improve solubilization

  • Consider using specialized plant protein extraction kits that address the unique challenges of plant tissues

How can I troubleshoot cross-reactivity issues with CYP71B35 antibodies against other CYP family members?

Cross-reactivity with related cytochrome P450 family members is a common challenge when working with CYP71B35 antibodies. To address this issue:

• Epitope analysis:

  • Perform bioinformatic analysis to identify unique regions in CYP71B35 compared to other CYP family members

  • Consider custom antibodies against these unique epitopes

• Specificity testing:

  • Test antibodies against recombinant proteins of closely related CYP family members (particularly other CYP71 subfamily members)

  • Perform Western blots with wild-type vs. CYP71B35 knockout/knockdown plant samples

• Antibody purification:

  • Consider affinity purification using recombinant CYP71B35 protein

  • Pre-absorb antibodies with recombinant proteins of related CYP family members to remove cross-reactive antibodies

• Alternative detection strategies:

  • Use multiple antibodies targeting different regions of CYP71B35

  • Combine antibody detection with mass spectrometry for definitive identification

A study investigating the specificity of research antibodies against NF-κB-subunit p65 found significant variation in specificity among commercially available antibodies, highlighting the importance of rigorous validation . Similar considerations apply to CYP71B35 antibodies, particularly given the high sequence homology among cytochrome P450 family members.

What are the best approaches for studying CYP71B35 protein-protein interactions?

To investigate protein-protein interactions involving CYP71B35:

• Co-immunoprecipitation (Co-IP):

  • Optimize lysis conditions to preserve native protein complexes

  • Use appropriate detergents for membrane protein solubilization

  • Consider crosslinking to stabilize transient interactions

  • Validate interactions with reciprocal Co-IP experiments

• Advanced interaction methods:

  • Apply proximity labeling techniques (BioID, APEX) for in vivo interaction mapping

  • Consider fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for in planta interaction studies

  • Use yeast two-hybrid screening with the soluble domains of CYP71B35

• Validation strategies:

  • Confirm interactions using orthogonal methods

  • Perform domain mapping to identify interaction interfaces

  • Examine co-localization using immunofluorescence microscopy

For a comprehensive approach, researchers have used Chromium Technology (10x Genomics) for single-cell encapsulation and sequencing to identify potential interaction partners, which could be adapted for CYP71B35 studies .

How can I use CYP71B35 antibodies to investigate changes in protein expression during plant stress responses?

To study CYP71B35 expression changes during plant stress responses:

• Experimental design:

  • Develop time-course experiments exposing plants to relevant stressors

  • Include proper controls (untreated plants, plants exposed to different stressors)

  • Consider tissue-specific sampling to identify localized responses

• Quantitative analysis methods:

  • Use quantitative Western blotting with appropriate loading controls

  • Consider ELISA for high-throughput quantification across multiple samples

  • Employ flow cytometry for single-cell level expression analysis

• Localization studies:

  • Perform immunohistochemistry to examine tissue-specific changes

  • Use immunofluorescence microscopy to track subcellular localization changes

• Multi-omics integration:

  • Correlate protein-level changes (using antibody-based methods) with transcriptomic data

  • Integrate with metabolomic analyses to identify changes in substrates/products

• Post-translational modification analysis:

  • Investigate potential stress-induced modifications (phosphorylation, ubiquitination)

  • Consider using modification-specific antibodies if available

These approaches can provide a comprehensive understanding of how CYP71B35 expression and function respond to environmental stresses, potentially revealing its role in plant stress adaptation mechanisms.