CESA7 Antibody

What is CESA7 and why are CESA7 antibodies important in plant cell wall research?

CESA7 is a critical cellulose synthase protein involved in secondary cell wall formation in plants. It functions as part of a complex with CESA4 and CESA8 to form the cellulose synthase complex (CSC) in secondary cell walls. CESA7 antibodies are essential tools for detecting, localizing, and studying this protein's expression, allowing researchers to understand its role in cell wall development.

The importance of CESA7 is highlighted by studies showing that knockout mutants (such as the irx3-7 mutant) cannot be complemented by other CESA genes, demonstrating the non-redundant, class-specific function of this protein . CESA7 antibodies allow researchers to visualize and quantify this protein in various experimental contexts, making them indispensable for understanding cellulose synthesis mechanisms in plants.

How specific are commercially available CESA7 antibodies?

CESA7 antibodies are typically raised against the variable region 1 (VR1) domain, which exhibits high class specificity based on bioinformatic analyses of 82 CESA proteins across 11 plant species . This domain choice is strategic, as the VR1 region shows significant sequence diversity between different CESA classes, enabling antibodies to distinguish between CESA isoforms.

Experimental data demonstrates that CESA7 exhibits very high class specificity, particularly in the N-terminal region, which distinguishes it from other CESA proteins like CESA4 . When selecting a CESA7 antibody, researchers should verify the epitope region and confirm the antibody has been validated specifically for their plant species of interest.

What are the optimal storage conditions for maintaining CESA7 antibody activity?

While specific storage recommendations for CESA7 antibodies are not provided in the search results, standard antibody storage protocols can be applied. Based on comparable antibody information:

• Store unopened/unconstituted antibodies at -20°C to -70°C

• After reconstitution, store at 2-8°C for short-term use (up to 1 month)

• For long-term storage (up to 6 months), aliquot and store at -20°C to -70°C

• Avoid repeated freeze-thaw cycles as they can significantly reduce antibody activity

For optimal performance, always follow manufacturer recommendations for the specific CESA7 antibody product being used.

How can I determine if my CESA7 antibody cross-reacts with other CESA family members?

Cross-reactivity assessment is crucial for accurate interpretation of CESA7 antibody results. Implement these validation strategies:

• Negative controls using knockout mutants: Test your antibody in cesa7 knockout lines (e.g., irx3-7) to confirm absence of signal. Presence of signal would indicate cross-reactivity with other proteins .

• Epitope analysis: Compare sequence alignment of the antibody epitope region across CESA family members. Higher sequence similarity with other CESA proteins increases cross-reactivity risk.

• Western blot validation: Run protein extracts from wild-type plants alongside extracts from plants overexpressing specific CESA proteins. CESA7 typically appears at approximately 120-125 kDa.

• Pre-absorption control: Pre-incubate the antibody with purified CESA7 peptide before use in immunodetection to confirm specificity.

The class specificity analysis by Carroll and Specht (2011) indicated that CESA7 exhibits high class specificity, particularly in the N-terminal region, making antibodies targeting this region less likely to cross-react with other CESA family members .

What are the key considerations when using CESA7 antibodies for co-immunoprecipitation of the cellulose synthase complex?

When using CESA7 antibodies for co-immunoprecipitation (co-IP) of the cellulose synthase complex, consider these important factors:

• Antibody format selection: For co-IP applications, use antibodies that maintain binding efficiency under native conditions. Full IgG formats typically perform better than antibody fragments like Fabs or scFvs in pull-down experiments .

• Complex preservation: CESA7 functions in a complex with CESA4 and CESA8. Use gentle cell lysis buffers with mild detergents (0.5-1% NP-40 or Triton X-100) to maintain complex integrity.

• Antibody concentration optimization: Titrate antibody concentrations to maximize specific pull-down while minimizing non-specific binding. Start with 1-5 μg antibody per 100-500 μg total protein.

• Controls: Always include:

  • IgG isotype control to identify non-specific binding

  • Input sample (pre-IP) to confirm target protein presence

  • cesa7 mutant tissue as a negative control

• Validation of interaction partners: Confirm pulled-down complexes contain expected interaction partners (CESA4 and CESA8) using specific antibodies for these proteins.

Research demonstrates that CESA7 works in a non-redundant manner with CESA4 and CESA8, forming a specific complex essential for secondary cell wall formation .

How can contradictory CESA7 antibody results be interpreted when comparing different tissue types or developmental stages?

When facing contradictory CESA7 antibody results across different tissues or developmental stages, consider these analytical approaches:

• Expression regulation assessment: CESA7 expression is highly regulated during development. Confirm tissue-specific expression patterns using RT-qPCR to correlate transcript and protein levels.

• Post-translational modification analysis: CESA proteins undergo phosphorylation and other modifications that might affect antibody recognition . If antibodies target regions containing potential modification sites, results may vary depending on the protein's modification state in different tissues.

• Protein complex formation dynamics: The formation of the cellulose synthase complex with CESA4 and CESA8 might mask epitopes in certain contexts. Use multiple antibodies targeting different CESA7 regions to verify results.

• Technical validation:

  • Perform loading controls with housekeeping proteins specific to each tissue type

  • Use standardized protein extraction protocols optimized for each tissue

  • Include positive controls from tissues known to express CESA7 (vascular tissues)

• Orthogonal validation: Complement antibody-based detection with fluorescently tagged CESA7 constructs expressed under native promoters to independently verify localization patterns.

Comprehensive functional analysis studies show that CESA7 exhibits tissue-specific patterns corresponding to secondary cell wall formation, primarily in vascular tissues .

What are the optimal conditions for using CESA7 antibodies in Western blot applications?

For optimal Western blot detection of CESA7, follow these evidence-based recommendations:

• Sample preparation:

  • Extract proteins using buffer containing 1% SDS, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and protease inhibitors

  • Add phosphatase inhibitors if studying phosphorylation states

  • Heat samples at 70°C for 10 minutes rather than boiling to prevent aggregation

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal separation of CESA7 (approximately 120-125 kDa)

  • Include positive control samples from tissues known to express CESA7

• Transfer conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for CESA proteins)

  • Use wet transfer at 30V overnight at 4°C for more efficient transfer of large proteins

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk or BSA in TBST

  • Typical primary antibody dilution range: 1:1000-1:5000 (optimize for specific antibody)

  • Incubate primary antibody overnight at 4°C for maximum sensitivity

• Detection optimization:

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

  • For low abundance detection, consider enhanced chemiluminescence (ECL) substrate systems

For reference, when detecting similar-sized membrane proteins, conditions similar to those used for detecting Carboxylesterase 2/CES2 might be applicable: probing PVDF membranes with 1 μg/mL primary antibody followed by HRP-conjugated secondary antibody under reducing conditions .

How can I optimize immunolocalization of CESA7 in plant tissue sections?

For successful immunolocalization of CESA7 in plant tissues, follow this optimized protocol:

• Tissue fixation and embedding:

  • Fix tissues in 4% paraformaldehyde in PBS for 2-4 hours at room temperature

  • For woody tissues, consider brief vacuum infiltration to improve fixative penetration

  • Dehydrate through ethanol series and embed in paraffin or LR White resin for improved epitope preservation

• Section preparation:

  • Cut 5-10 μm sections for paraffin or 1-2 μm sections for resin

  • Mount on positively charged slides

  • For paraffin sections: deparaffinize with xylene and rehydrate through ethanol series

• Antigen retrieval (critical for formalin-fixed tissues):

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 10-20 minutes)

• Blocking and antibody incubation:

  • Block with 5% normal serum (corresponding to secondary antibody host) with 0.3% Triton X-100

  • Incubate with primary CESA7 antibody (1:100-1:500) overnight at 4°C

  • Wash thoroughly and incubate with fluorophore-conjugated secondary antibody (1:200-1:500)

• Controls and validation:

  • Include cesa7 mutant tissue sections as negative controls

  • Perform peptide competition assay by pre-incubating antibody with immunizing peptide

  • Consider dual labeling with markers for specific cell types or subcellular compartments

Research shows that CESA7 localizes primarily to vascular tissues undergoing secondary cell wall formation, so targeting these regions during tissue selection will increase success rates .

What approaches can be used to quantify CESA7 protein levels in different plant genotypes or conditions?

For accurate quantification of CESA7 protein levels across experimental conditions, employ these validated approaches:

• Quantitative Western blotting:

  • Use internal loading controls (anti-actin or anti-tubulin)

  • Include a standard curve of purified recombinant CESA7 protein

  • Analyze band intensity using software like ImageJ

  • Perform at least three biological replicates for statistical analysis

  Sample Type	Normalization Method	Detection Range	Best For
  Crude extract	Total protein (Ponceau)	10-100 ng	Relative comparisons
  Membrane fraction	Membrane marker proteins	5-50 ng	Enriched samples
  Immunoprecipitated	IgG heavy chain	1-20 ng	Interacting partners

• ELISA-based quantification:

  • Develop sandwich ELISA using two antibodies targeting different CESA7 epitopes

  • Create standard curves using purified CESA7 protein

  • Optimize extraction buffers to solubilize membrane-bound CESA7

• Mass spectrometry approaches:

  • Use selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Identify CESA7-specific peptides for targeted quantification

  • Include isotopically labeled peptide standards for absolute quantification

• Flow cytometry (for protoplasts or cell suspensions):

  • Permeabilize cells and perform intracellular staining with CESA7 antibodies

  • Use fluorophore-conjugated secondary antibodies

  • Quantify signal intensity across cell populations

• Image analysis of immunolocalization:

  • Maintain identical acquisition settings across samples

  • Quantify fluorescence intensity in defined cellular regions

  • Use appropriate statistical tests for comparing conditions

When comparing mutants, it's important to note that functional analysis studies have shown that cesa4 irx5-4, cesa7 irx3-7, and cesa8 irx1-7 mutants exhibit defects in cellulose content that can only be complemented by their respective wild-type genes, highlighting the non-redundant nature of these proteins .

How can I address weak or absent signals when using CESA7 antibodies in Western blots?

When encountering weak or absent CESA7 signals in Western blots, implement this systematic troubleshooting approach:

• Protein extraction optimization:

  • CESA7 is a membrane-bound protein; use extraction buffers containing 1-2% SDS or 8M urea

  • Include protease inhibitors to prevent degradation

  • Avoid excessive heating which may cause protein aggregation

  • Consider enriching for membrane fractions before analysis

• Antibody-specific factors:

  • Verify antibody viability with a positive control sample

  • Test increased antibody concentration (2-5× standard dilution)

  • Extend primary antibody incubation to overnight at 4°C

  • Check antibody storage conditions and expiration date

• Transfer efficiency assessment:

  • Verify transfer of high molecular weight proteins using Ponceau S staining

  • Increase transfer time or use specialized transfer systems for large proteins

  • Consider using PVDF membrane instead of nitrocellulose for better retention

• Detection system enhancement:

  • Use more sensitive ECL substrates for HRP-conjugated antibodies

  • Consider signal amplification systems (biotin-streptavidin)

  • Increase exposure time during imaging

• Biological considerations:

  • Confirm CESA7 expression in your experimental tissue/conditions

  • Use tissues undergoing active secondary cell wall formation

  • Consider developmental timing as CESA7 expression is highly regulated

Research has demonstrated that CESA7 is primarily expressed in tissues undergoing secondary cell wall formation, so samples should be collected from appropriate developmental stages and tissues .

What factors might contribute to non-specific binding of CESA7 antibodies and how can they be mitigated?

Non-specific binding is a common challenge with antibodies targeting plant proteins. Address CESA7 antibody non-specificity with these strategies:

• Optimization of blocking conditions:

  • Test different blocking agents (5% milk, 5% BSA, commercial blocking buffers)

  • Extend blocking time to 2-3 hours at room temperature

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

• Antibody dilution optimization:

  • Perform antibody titration to identify optimal concentration

  • Prepare antibody dilutions in blocking buffer containing 0.05-0.1% Tween-20

  • Consider adding 1-5% of the blocking agent to antibody dilution buffer

• Stringency adjustment in wash steps:

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

  • Add 0.1-0.5% Tween-20 or 0.1% Triton X-100 to wash buffer

  • Extend and increase number of wash steps (5-6 washes, 10 minutes each)

• Pre-absorption techniques:

  • Pre-incubate antibody with protein extract from cesa7 knockout plants

  • For polyclonal antibodies, consider affinity purification against the immunizing peptide

• Epitope consideration:

  • Antibodies targeting highly conserved regions (CR1, CR2) may cross-react with other CESA proteins

  • Prefer antibodies targeting class-specific regions like VR1, which shows high specificity for CESA7

Research has shown that CESA7 exhibits high class specificity in multiple regions, particularly in the N-terminal region and variable regions (VR1 and VR2), which can be leveraged to develop more specific antibodies .

How can CESA7 antibodies be used to study protein-protein interactions within the cellulose synthase complex?

CESA7 antibodies provide powerful tools for investigating protein-protein interactions within the cellulose synthase complex through these advanced methods:

• Co-immunoprecipitation (Co-IP) coupled with mass spectrometry:

  • Use CESA7 antibodies to pull down intact complexes

  • Analyze by LC-MS/MS to identify interaction partners

  • Compare wild-type vs. mutant backgrounds to identify condition-specific interactions

  • Quantify relative abundance of CESA4, CESA7, and CESA8 in the complex

• Proximity-dependent labeling:

  • Generate fusion constructs of CESA7 with BioID or APEX2

  • Use CESA7 antibodies to confirm expression and localization

  • Identify proximal proteins through streptavidin pull-down and mass spectrometry

• Förster Resonance Energy Transfer (FRET) analysis:

  • Perform immunolabeling with CESA7 antibodies and fluorophore-conjugated secondary antibodies

  • Label potential interaction partners with complementary fluorophores

  • Measure FRET efficiency to assess protein proximity

• In situ Proximity Ligation Assay (PLA):

  • Use CESA7 antibodies in combination with antibodies against potential interacting partners

  • Generate fluorescent signals only when proteins are in close proximity (<40 nm)

  • Quantify interaction events through fluorescent spot analysis

• Sequential immunoprecipitation:

  • Perform first IP with CESA7 antibodies

  • Elute complexes and perform second IP with antibodies against CESA4 or CESA8

  • Analyze doubly-purified complexes to confirm direct interactions

Functional analysis studies have confirmed that CESA7 works in a complex with CESA4 and CESA8, with each protein playing a non-redundant role in secondary cell wall formation .

What modifications can be made to CESA7 antibodies to enhance their specificity or functionality for specialized applications?

For specialized applications requiring enhanced CESA7 antibody performance, consider these advanced modifications:

• Antibody fragment generation:

  • Create Fab or F(ab')₂ fragments by enzyme digestion to reduce non-specific binding

  • Generate single-chain variable fragments (scFv) for applications requiring smaller probe size

  • Use nanobodies (VHH) for enhanced tissue penetration and epitope accessibility

• Affinity maturation:

  • Perform in vitro affinity maturation through display technologies

  • Select higher-affinity variants through competitive binding assays

  • Engineer complementarity-determining regions (CDRs) for improved specificity

• Conjugation strategies:

  • Direct conjugation to fluorophores for single-step immunodetection

  • Attach gold nanoparticles for electron microscopy applications

  • Conjugate to biotin or digoxigenin for signal amplification systems

• Cross-linking modifications:

  • Introduce photo-activatable crosslinkers to capture transient interactions

  • Add chemical crosslinkers to stabilize antibody-antigen complexes

• Bispecific antibody construction:

  • Engineer bispecific antibodies targeting CESA7 and another CSC component

  • Create formats like diabodies or dual-variable-domain antibodies

  • Use these to study complex assembly or for enhanced avidity

Research in antibody engineering demonstrates that variable region modifications can significantly improve specificity and functionality, with formats like scFv providing more freedom for modification than full immunoglobulins .