IBR5 Antibody

What is IBR5 and why are antibodies against it important for research?

IBR5 encodes a dual-specificity MAPK phosphatase that functions as a positive regulator of plant responses to auxin and ABA. Recent research has revealed its critical role in plant immune responses, particularly in temperature-dependent defense mechanisms . IBR5 antibodies are essential research tools for:

• Detecting and quantifying IBR5 protein levels in different tissues and conditions

• Investigating protein-protein interactions, particularly with immune receptors like CHS3

• Studying the role of IBR5 in R protein-mediated defense responses

• Examining how IBR5 contributes to temperature stress responses

• Exploring its function in hormone signaling pathways

Studies have demonstrated that IBR5 protein accumulates significantly in the chs3-1 mutant, suggesting it plays a role in stabilizing the CHS3 protein . Specific antibodies against IBR5 are therefore crucial for detecting these changes in protein abundance and understanding underlying mechanisms.

What validation methods should I use to confirm IBR5 antibody specificity?

Proper validation is critical for ensuring experimental reliability when working with IBR5 antibodies:

Western blot validation:

• Compare wild-type samples with ibr5 mutants (such as ibr5-3 or ibr5-7)

• Look for a band of expected molecular weight that is absent or reduced in mutant samples

• Liu et al. demonstrated this approach by showing IBR5 protein presence in wild-type plants, reduced levels in ibr5-7, and complete absence in ibr5-3

Immunoprecipitation validation:

• Perform IP with the IBR5 antibody followed by Western blot analysis

• Confirm the immunoprecipitated protein is recognized by other IBR5 antibodies

• Verify protein identity through mass spectrometry when necessary

Genetic validation:

• Use complementation lines expressing IBR5 in ibr5 mutant backgrounds

• Confirm antibody signal reappears in these complementation lines

• Use inducible expression systems to show correlation between gene expression and antibody signal

What controls should be included when working with IBR5 antibodies?

Based on established research practices and the available literature, include:

Positive controls:

• Wild-type plant samples known to express IBR5

• Recombinant IBR5 protein

• Tissues from plants overexpressing IBR5 (such as lines with 35S:HA-Flag-IBR5)

Negative controls:

• IBR5 knockout or null mutant samples (ibr5-3 shows no detectable IBR5 protein)

• Pre-immune serum (for polyclonal antibodies)

• Isotype controls (for monoclonal antibodies)

• Secondary antibody-only controls

The study by Liu et al. effectively used the ibr5-3 mutant as a negative control, demonstrating complete absence of IBR5 protein, while the ibr5-7 mutant showed reduced IBR5 levels compared to wild-type .

How should IBR5 antibodies be stored and handled?

While specific storage information for IBR5 antibodies is not detailed in the search results, standard antibody handling practices should be followed:

Storage recommendations:

• Store at -20°C for long-term storage

• For working solutions, maintain at 4°C for up to one month

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Consider adding glycerol (final concentration 30-50%) for stability

Handling guidelines:

• Centrifuge antibody vials briefly before opening

• Use sterile techniques when handling antibody solutions

• Mix gently by inversion or gentle pipetting rather than vortexing

• Allow refrigerated antibodies to equilibrate to room temperature before opening

What expression patterns of IBR5 should I expect in different plant tissues?

Based on the available research:

• IBR5 is expressed in various plant tissues but shows differential accumulation under stress conditions

• Accumulation significantly increases in the chs3-1 mutant, suggesting regulation during defense responses

• Expression may vary in response to temperature changes, particularly chilling conditions

• IBR5 levels should be examined in both normal growth conditions and under specific stresses

Researchers should be prepared for potential variations in IBR5 expression levels depending on:

• Plant developmental stage

• Tissue type

• Environmental conditions (particularly temperature)

• Presence of pathogens or activation of defense responses

How can IBR5 antibodies be used to study plant temperature-dependent immune responses?

IBR5 antibodies are valuable tools for investigating temperature-sensitive immune pathways, particularly in the context of chilling sensitivity as described by Liu et al. :

Protein accumulation analysis:

• Use Western blotting with IBR5 antibodies to monitor changes in IBR5 protein levels at different temperatures

• Compare wild-type plants with temperature-sensitive mutants like chs3-1

• Quantify IBR5 abundance during temperature shifts and correlate with defense marker expression

Protein complex dynamics:

• Employ co-immunoprecipitation with IBR5 antibodies to isolate protein complexes under varying temperatures

• Analyze how temperature affects interactions between IBR5 and partners like CHS3, HSP90, and SGT1b

• Determine if cold treatment alters the composition or stability of these complexes

Liu et al. demonstrated that IBR5 forms a complex with CHS3, HSP90, and SGT1b that protects CHS3, particularly important under chilling conditions . Using IBR5 antibodies, researchers can further investigate how this protective complex responds to temperature fluctuations.

What are the optimal protocols for using IBR5 antibodies in co-immunoprecipitation studies?

Based on approaches described in the literature :

Sample preparation:

• Use freshly prepared plant tissue harvested under specific conditions (temperature, pathogen exposure)

• Extract proteins in buffers that preserve native interactions (containing mild detergents)

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

Immunoprecipitation procedure:

• Incubate cleared lysates with optimized amounts of IBR5 antibody

• Capture antibody-protein complexes with protein A/G beads

• Wash extensively to remove non-specifically bound proteins

• Elute bound proteins for analysis by Western blotting or mass spectrometry

Experimental design:

• Perform reciprocal co-IPs using both IBR5 antibodies and antibodies against suspected interaction partners

• For transiently expressed proteins, use epitope tags as demonstrated with HA-Flag-IBR5 and Myc-tagged proteins

• Include appropriate controls: IgG control, input samples, and lysates from mutant plants

Liu et al. successfully demonstrated IBR5-CHS3 interactions using co-IP approaches in both Arabidopsis protoplasts and N. benthamiana, showing that IBR5 interacted specifically with the TIR domain of CHS3 .

How can I use IBR5 antibodies to study its dual roles in defense and hormone signaling?

The search results indicate that IBR5 functions in both defense responses and auxin signaling pathways, with some evidence of separable roles :

Comparative protein interaction analysis:

• Use IBR5 antibodies to immunoprecipitate protein complexes from plants treated with pathogens versus auxin

• Compare the interactomes to identify shared versus pathway-specific interaction partners

• Analyze how mutations in specific domains affect these distinct interactions

Functional domain studies:

• Use domain-specific IBR5 antibodies to determine which regions mediate different interactions

• Compare wild-type IBR5 with the catalytically inactive IBR5^C129S to determine role of phosphatase activity

• Liu et al. showed IBR5^C129S only partially rescued chs3 ibr5 phenotypes, indicating phosphatase activity is important but not the only function

Pathway-specific activity assays:

• Immunoprecipitate IBR5 from plants under different conditions using specific antibodies

• Test phosphatase activity against different substrates (MPK12 for auxin signaling; potential defense pathway substrates)

• Determine if IBR5's catalytic activity changes during defense versus hormone responses

What approaches can reveal the molecular mechanism of IBR5's holdase activity?

Liu et al. reported that IBR5 exhibits holdase activity and physically associates with CHS3, HSP90 and SGT1b to form a protective complex :

Protein stability assays:

• Use IBR5 antibodies to monitor CHS3 protein levels in wild-type versus ibr5 mutant plants

• Perform pulse-chase experiments to examine protein turnover rates

• Test whether IBR5's holdase function is separable from its phosphatase activity

Complex assembly analysis:

• Employ size exclusion chromatography followed by Western blotting with IBR5 antibodies

• Determine the stoichiometry and order of assembly of the IBR5-CHS3-HSP90-SGT1b complex

• Identify which domains of IBR5 are critical for holdase versus phosphatase functions

Chaperone activity assays:

• Use purified components and IBR5 antibodies to monitor protein aggregation

• Test whether IBR5 prevents thermal denaturation of client proteins like CHS3

• Compare holdase activity of wild-type IBR5 versus phosphatase-dead IBR5^C129S

How might IBR5 antibodies contribute to understanding broader R-protein mediated immunity?

The search results indicate that IBR5 functions in multiple R protein-mediated defense pathways beyond CHS3, including SNC1, RPS4, and RPM1 :

Comparative immunoprecipitation:

• Use IBR5 antibodies to pull down protein complexes from plants expressing different R proteins

• Identify common versus R protein-specific components of these complexes

• Determine if IBR5 interacts directly with multiple R proteins or via shared adaptor proteins

R-protein stabilization analysis:

• Monitor R protein levels using specific antibodies in wild-type versus ibr5 mutant backgrounds

• Test whether IBR5's effect on R protein stability is universal or specific to certain classes

• Examine how environmental conditions affect these stabilization functions

Defense output measurements:

• Correlate IBR5 protein levels (detected via antibodies) with expression of defense marker genes

• Compare defense outputs across different R protein activation scenarios with or without functional IBR5

• Determine whether IBR5's role is specific to certain defense signaling branches

Table 1: Comparison of Methods Using IBR5 Antibodies for Protein Detection

Table 2: IBR5 Protein Interactions Detectable Using Co-Immunoprecipitation with IBR5 Antibodies

What factors might affect IBR5 antibody specificity in plant samples?

When working with plant samples, several factors can influence IBR5 antibody performance:

Sample preparation challenges:

• Plant tissues contain abundant polyphenols and other compounds that can interfere with antibody binding

• Extraction buffers should include PVPP, PVP, or other additives to remove these interfering compounds

• Protein degradation during extraction can reduce detectable IBR5 signal

Potential cross-reactivity:

• IBR5 belongs to the dual-specificity phosphatase family, which may share conserved domains

• Validate antibody specificity using ibr5 null mutants like ibr5-3 as demonstrated by Liu et al.

• Consider using epitope-tagged versions of IBR5 with tag-specific antibodies for highest specificity

Recommended solutions:

• Optimize extraction procedures for your specific plant tissue

• Use freshly prepared samples whenever possible

• Include protease inhibitors during all steps of protein isolation

• Validate antibodies in your specific experimental system before proceeding to complex experiments

How might advanced antibody technologies enhance IBR5 research?

Emerging technologies offer new opportunities for studying IBR5 function:

Single-cell antibody-based approaches:

• Single-cell Western blotting could reveal cell-to-cell variability in IBR5 expression

• Flow cytometry with intracellular staining (similar to approaches described in reference ) could analyze IBR5 expression patterns across cell populations

Humanized antibody development:

• Approaches similar to those described for rabbit monoclonal antibodies could be applied to generate high-affinity IBR5 antibodies

• These might offer improved specificity and sensitivity for detecting low-abundance IBR5 protein

Structure-guided antibody engineering:

• Similar to methodologies described in reference , structure-based approaches could generate antibodies specific to active versus inactive IBR5 conformations

• This would allow researchers to monitor IBR5 activation states in different signaling contexts

What emerging applications might benefit from IBR5 antibodies?

Climate resilience research:

• Given IBR5's role in temperature-dependent defense responses , antibodies could help elucidate mechanisms of plant adaptation to changing climates

• Monitoring IBR5-CHS3 complex formation under varying temperature regimes might reveal temperature sensing mechanisms

Synthetic immunity engineering:

• Understanding how IBR5 stabilizes multiple R proteins could inform efforts to engineer broad-spectrum disease resistance

• Antibodies would be essential tools for monitoring the stability and function of engineered immune complexes

Systems biology approaches:

• Integration of proteomic data (using IBR5 antibodies) with transcriptomic, metabolomic, and phenotypic datasets

• This could provide a comprehensive view of IBR5's role in coordinating defense and development