CYP705A20 Antibody

What is CYP705A20 and what is its function in Arabidopsis thaliana?

CYP705A20 (Entrez Gene ID: 821554) is a protein-coding gene in Arabidopsis thaliana (thale cress) that encodes cytochrome P450, family 705, subfamily A, polypeptide 20. It belongs to the large cytochrome P450 superfamily of enzymes, which are involved in the oxidation of various substrates in plants. This particular CYP is located at locus At3g20110 and is also known by the identifier MAL21.15 . The specific biochemical function of CYP705A20 is not fully characterized, but like other plant cytochrome P450s, it likely plays a role in secondary metabolism pathways, potentially involved in the synthesis of specialized metabolites that contribute to plant defense mechanisms or development.

What types of CYP705A20 antibodies are currently available for research?

Current research indicates availability of polyclonal antibodies against CYP705A20. Specifically, rabbit polyclonal antibodies against Arabidopsis thaliana CYP705A20 have been developed for research applications . These antibodies are typically purified using antigen-affinity methods to ensure specificity. According to the available information, these antibodies are designed to recognize the native CYP705A20 protein in Arabidopsis thaliana samples. The antibodies are typically characterized by their host species (rabbit), reactivity (Arabidopsis thaliana), and purification method (antigen-affinity) .

What are the validated applications for CYP705A20 antibodies?

CYP705A20 antibodies have been validated for several experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of CYP705A20 protein in plant extracts

• Western Blot (WB): For identification and semi-quantitative analysis of CYP705A20 in protein samples

These applications allow researchers to detect the presence of CYP705A20 protein in various experimental contexts, including protein expression studies, localization experiments, and comparative analyses across different plant tissues or developmental stages.

How should samples be prepared for optimal CYP705A20 detection?

Proper sample preparation is crucial for successful CYP705A20 detection. For Arabidopsis thaliana samples, the following methodology is recommended:

• Tissue collection: Harvest fresh plant tissue (leaves, roots, etc.) and immediately flash-freeze in liquid nitrogen to preserve protein integrity.

• Homogenization: Grind frozen tissue to a fine powder using a mortar and pestle under liquid nitrogen conditions.

• Protein extraction: Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail (to prevent protein degradation)

• Centrifugation: Clear the lysate by centrifugation at 10,000 × g for 15 minutes at 4°C.

• Protein quantification: Determine protein concentration using standard methods (Bradford, BCA, etc.).

This preparation method ensures that the CYP705A20 protein maintains its native conformation and epitope accessibility for antibody recognition in subsequent applications .

How can Western blot protocols be optimized for CYP705A20 detection?

For optimal detection of CYP705A20 in Western blot applications, consider the following protocol optimizations:

• Protein loading: Load 20-40 μg of total protein per lane for standard detection.

• Gel selection: Use 10-12% polyacrylamide gels, as CYP705A20 has a molecular weight consistent with other cytochrome P450 proteins (approximately 50-60 kDa).

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 100V for 1 hour in cold room (4°C)

  • Use PVDF membrane for optimal protein binding

• Blocking: Block with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody: Dilute anti-CYP705A20 antibody at 1:1000 to 1:2000 in blocking solution. Incubate overnight at 4°C.

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 to 1:10000 dilution for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence (ECL) for detection.

• Controls: Always include positive control (recombinant CYP705A20 protein) and negative control (extract from plants with CYP705A20 knockout) .

When troubleshooting, consider adjusting the antibody concentration, incubation time, or washing stringency based on signal-to-noise ratio.

What are best practices for validating CYP705A20 antibody specificity?

Validating antibody specificity is crucial for obtaining reliable research results. For CYP705A20 antibodies, implement these validation strategies:

• Recombinant protein control: Use purified recombinant CYP705A20 protein as a positive control to confirm antibody recognition of the target protein.

• Knockout/knockdown validation: Compare antibody reactivity between wild-type plants and those with CYP705A20 knockout/knockdown to confirm specificity.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to samples. Signal reduction indicates specificity.

• Cross-reactivity assessment: Test the antibody against other related cytochrome P450 family members, particularly those in the CYP705 subfamily, to assess potential cross-reactivity.

• Protein chip technology: Utilize Arabidopsis protein chip technology to evaluate antibody specificity against multiple proteins simultaneously. This approach can be particularly effective as demonstrated in studies with other plant antibodies, where researchers showed that monoclonal antibodies bound specifically to their respective antigens without cross-reacting with other proteins on the chips .

These validation steps ensure that experimental observations are attributable to CYP705A20 rather than non-specific binding or cross-reactivity with related proteins.

How can quantitative flow cytometry be adapted for CYP705A20 studies?

While not directly described for CYP705A20, flow cytometry techniques can be adapted from other antibody internalization studies:

• Sample preparation:

  • Harvest plant protoplasts or cultured plant cells

  • Wash cells in appropriate buffer (PBS with 0.1% BSA)

  • Fix cells using 2-4% paraformaldehyde if studying localization

• Labeling protocol:

  • Incubate cells with fluorophore-conjugated CYP705A20 antibody (such as Alexa Fluor 488 or 594 conjugates)

  • Wash to remove unbound antibody

  • For internalization studies, allow cells to incubate at 37°C for defined time intervals

• Surface quenching approach:

  • To differentiate between surface-bound and internalized antibodies, utilize anti-Alexa Fluor quenching antibodies

  • Anti-Alexa Fluor mAbs specifically quench cell surface fluorescence without affecting internalized fluorescence

• Data acquisition parameters:

  • Collect minimum 10,000 events per sample

  • Use appropriate laser excitation for the fluorophore (e.g., 488 nm for Alexa Fluor 488)

  • Establish proper gating strategies to exclude cell debris and aggregates

• Analysis:

  • Calculate the median fluorescence intensity for viable cells

  • For internalization studies, compare quenched vs. non-quenched samples to determine internalization rates

This methodology allows quantitative assessment of CYP705A20 expression and localization dynamics in plant cells.

What techniques can be used to study CYP705A20 localization in plant cells?

Several complementary techniques can be employed to study CYP705A20 subcellular localization:

• Immunofluorescence microscopy:

  • Fix plant tissue sections or protoplasts with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 3% BSA in PBS

  • Incubate with CYP705A20 antibody (1:100-1:500 dilution)

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain with DAPI for nuclei visualization

  • Image using confocal microscopy

• Subcellular fractionation combined with Western blotting:

  • Prepare and separate cellular fractions (cytosolic, microsomal, etc.)

  • Run Western blots on each fraction

  • Detect CYP705A20 using specific antibodies

  • Include markers for different cellular compartments as controls

• Dual-label immunofluorescence:

  • Co-stain with CYP705A20 antibody and markers for specific cellular compartments

  • For example, use anti-LAMP1 for lysosomes or Alexa Fluor 647-Phalloidin for actin cytoskeleton

  • Analyze colocalization using appropriate software

• Live-cell imaging with fluorescently-tagged antibodies:

  • Label CYP705A20 antibodies with Alexa Fluor dyes

  • Apply to live plant protoplasts or tissue

  • Monitor localization in real-time

This multi-faceted approach provides comprehensive information about CYP705A20's spatial distribution and potential functional compartmentalization within plant cells.

How can non-specific binding be reduced when using CYP705A20 antibodies?

Non-specific binding can significantly compromise experimental results. To minimize this issue:

• Blocking optimization:

  • Test different blocking agents: 5% non-fat dry milk, 3-5% BSA, or commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

• Antibody dilution:

  • Titrate primary antibody (try 1:500, 1:1000, 1:2000, 1:5000)

  • Determine optimal concentration that maximizes specific signal while minimizing background

• Washing stringency:

  • Increase number of washes (5-6 times for 5-10 minutes each)

  • Add higher concentrations of detergent to wash buffer (up to 0.3% Tween-20)

• Cross-adsorption:

  • Pre-adsorb antibody with plant extract from CYP705A20 knockout plants

  • This removes antibodies that bind to non-CYP705A20 epitopes

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration if background remains high

• Buffer optimization:

  • Add 0.1-0.5% BSA to antibody dilution buffers

  • Consider adding 0.1% gelatin or 0.05% sodium azide to reduce non-specific binding

These strategies help ensure that signals detected in experiments represent genuine CYP705A20 rather than artifacts from non-specific interactions .

What are the critical controls for CYP705A20 antibody experiments?

Proper controls are essential for the interpretation of results in CYP705A20 antibody experiments:

• Positive controls:

  • Recombinant CYP705A20 protein (full-length or partial)

  • Tissues known to express high levels of CYP705A20

  • Transgenic plants overexpressing CYP705A20

• Negative controls:

  • CYP705A20 knockout or knockdown plant tissues

  • Tissues known not to express CYP705A20

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control (irrelevant antibody of same isotype and concentration)

• Specificity controls:

  • Peptide competition/blocking experiments

  • Pre-immune serum control (for polyclonal antibodies)

  • Cross-reactivity testing with related CYP proteins

• Technical controls:

  • Loading controls (e.g., anti-actin or anti-tubulin antibodies)

  • Markers for subcellular compartments in localization studies

  • Concentration gradients to establish assay linearity

Implementation of these controls enables confident interpretation of experimental results and helps distinguish genuine biological findings from technical artifacts.

How can protein chip technology be utilized for CYP705A20 research?

Protein chip technology offers powerful capabilities for high-throughput analysis of CYP705A20 interactions and function:

• Chip preparation:

  • Express and purify recombinant RGS-His6-tagged CYP705A20 protein

  • Robotically array proteins onto appropriate slides (FAST slides with nitrocellulose-based polymer or PAA slides with polyacrylamide)

  • Detection limits vary by substrate: approximately 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides

• Applications:

  • Antibody characterization: Test specificity of anti-CYP705A20 antibodies

  • Protein-protein interaction studies: Identify proteins that interact with CYP705A20

  • Substrate specificity analysis: Screen potential substrates for enzymatic activity

• Experimental workflow:

  • Print purified CYP705A20 alongside other proteins as controls

  • Probe with antibodies, potential interacting proteins, or substrate mixtures

  • Detect binding using fluorescent or colorimetric methods

  • Analyze results to identify specific interactions

• Technical considerations:

  • Use anti-RGS-His6 antibody to verify successful protein printing

  • Include appropriate positive and negative controls on each chip

  • Perform replicate experiments to ensure reproducibility

This technology allows systematic screening of CYP705A20 interactions at a scale not possible with traditional biochemical methods, enabling discovery of novel functions and regulatory mechanisms .

What are the considerations for dual-labeling experiments with CYP705A20 antibodies?

Dual-labeling experiments allow simultaneous detection of CYP705A20 and other proteins of interest:

• Antibody selection:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-CYP705A20 and mouse anti-second protein)

  • Ensure antibodies have been validated for the intended application

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap (e.g., Alexa Fluor 488 and Alexa Fluor 594)

  • Consider using anti-Alexa Fluor antibodies for specific quenching in internalization studies

• Protocol considerations:

  • Apply primary antibodies sequentially or simultaneously (test both to optimize)

  • Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity

  • Include single-label controls to verify specificity

• Analysis approach:

  • Use appropriate filter sets during microscopy to prevent bleed-through

  • Apply colocalization analysis software to quantify spatial relationships

  • Consider time-resolved imaging for dynamic processes

• Potential applications:

  • Colocalization of CYP705A20 with organelle markers

  • Investigation of protein-protein interactions in situ

  • Analysis of sequential protein recruitment during cellular processes

When properly implemented, dual-labeling experiments provide valuable insights into the spatial and functional relationships between CYP705A20 and other cellular components .

How can binding kinetics of CYP705A20 antibodies be determined?

Understanding antibody binding kinetics is crucial for optimizing experimental conditions. Several approaches can be used:

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant CYP705A20 on a sensor chip

  • Flow antibody solutions over the chip at different concentrations

  • Measure association and dissociation rates in real-time

  • Calculate affinity constants (KD, ka, kd)

• Bio-Layer Interferometry (BLI):

  • Similar to the approach used for other antibodies in the search results

  • Immobilize anti-CYP705A20 antibodies onto biosensor surfaces

  • Measure binding to titrated concentrations of recombinant CYP705A20 protein

  • Analyze data using a 1:1 Langmuir binding model

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Coat plates with recombinant CYP705A20

  • Apply antibody at various concentrations

  • Develop using appropriate secondary antibody and substrate

  • Plot binding curve and calculate apparent KD

• Flow Cytometry:

  • Incubate cells expressing CYP705A20 with different antibody concentrations

  • Measure median fluorescence intensity

  • Calculate apparent affinity from the resulting binding curve

These measurements help optimize antibody concentration for experiments and compare different antibody preparations .

How does CYP705A20 relate to other cytochrome P450 proteins in plants?

CYP705A20 belongs to the diverse cytochrome P450 superfamily in plants, with specific characteristics:

• Evolutionary context:

  • Member of the CYP705 family, which is specific to the Brassicaceae plant family

  • Part of the larger CYP71 clan of plant P450s involved in specialized metabolism

  • Evolved from ancestral CYP genes through gene duplication and functional diversification

• Structural features:

  • Contains conserved domains typical of P450 enzymes, including the heme-binding domain

  • Likely membrane-bound, associated with the endoplasmic reticulum

  • Substrate recognition sites show divergence from other CYP705 family members

• Functional relationships:

  • May participate in triterpenoid biosynthesis pathways

  • Potentially involved in plant defense mechanisms

  • Could function in coordination with other enzymes in metabolic pathways

• Comparative expression:

  • Expression patterns may differ from other CYP705 family members

  • Tissue-specific or stress-induced expression provides clues to function

  • Co-expression with other genes can indicate functional relationships

Understanding these relationships provides context for interpreting CYP705A20 research findings and suggests directions for future investigations.

What bioinformatic approaches can enhance CYP705A20 antibody research?

Bioinformatic analysis can significantly strengthen experimental design and interpretation:

• Epitope prediction:

  • Use algorithms to identify likely antigenic regions of CYP705A20

  • Assess conservation of epitopes across related proteins

  • Predict potential cross-reactivity with other plant proteins

• Structural modeling:

  • Generate homology models of CYP705A20 based on related P450 structures

  • Map epitope locations on the 3D structure

  • Predict accessibility of epitopes in native protein

• Expression analysis:

  • Mine transcriptomic databases for CYP705A20 expression patterns

  • Identify conditions that upregulate or downregulate expression

  • Guide tissue selection for maximum protein detection

• Sequence comparison:

  • Align CYP705A20 with related proteins to identify unique regions

  • Design experiments to test antibody specificity against close homologs

  • Predict potential post-translational modifications that might affect antibody binding

• Network analysis:

  • Identify proteins that may interact with CYP705A20

  • Suggest candidates for co-immunoprecipitation experiments

  • Place CYP705A20 in broader metabolic or signaling pathways

These bioinformatic approaches provide valuable context for experimental design and help researchers interpret their findings within the broader biological framework of plant metabolism and physiology.