CHI-B Antibody

What is CHI-B Antibody and what is its target protein?

CHI-B antibody (product code CSB-PA324776XA01DOA) is a research antibody that recognizes and binds to the chitinase-B (CHI-B) protein from Arabidopsis thaliana (Mouse-ear cress), which has the UniProt accession number P19171 . CHI-B is an endochitinase that catalyzes the hydrolytic cleavage of the β-1,4-N-acetyl-D-glucosamine linkages in chitin polymers. This enzyme plays a key role in plant defense mechanisms against fungal pathogens, as chitin is a major component of fungal cell walls.

How does CHI-B differ from other chitinases?

While the search results don't specify the exact differences between CHI-B and other chitinases in Arabidopsis, we can draw comparisons with other chitinase systems. For example, in the marine bacterium Alteromonas sp. strain O-7, four distinct chitinases (ChiA, ChiB, ChiC, and ChiD) have been identified, each with different substrate preferences and activities . Similar to this bacterial system, plant chitinases like CHI-B typically have specific domains including a catalytic domain, a chitin-binding domain, and possibly other structural elements that contribute to their function and specificity .

What is the structural composition of CHI-B protein?

Based on information about chitinases from other systems, we can infer that CHI-B likely contains a catalytic domain responsible for enzymatic activity and a chitin-binding domain that facilitates interaction with its substrate . Chitinases typically belong to glycosyl hydrolase family 18 or 19, with plant chitinases most commonly belonging to family 19 . The specific domain architecture of CHI-B in Arabidopsis would need to be confirmed through structural studies or database information from resources like UniProt.

What are the primary research applications for CHI-B antibody?

CHI-B antibody is primarily used for detecting and quantifying CHI-B protein expression in Arabidopsis thaliana samples. This can be particularly valuable in studying:

• Plant immune responses to fungal pathogens

• Developmental regulation of chitinase expression

• Effects of environmental stressors on defense protein production

• Protein localization studies using immunohistochemistry or immunofluorescence techniques

How can CHI-B antibody help in studying plant defense mechanisms?

Chitinases like CHI-B are key components of plant innate immunity against fungal pathogens. The CHI-B antibody allows researchers to monitor changes in CHI-B protein levels during pathogen challenge, providing insights into the timing and magnitude of defense responses. By analogy with bacterial chitinase systems, where multiple chitinases work synergistically in chitin degradation , studying CHI-B in plants can reveal how different chitinases cooperate in plant defense. Researchers can use the antibody to determine whether CHI-B expression is constitutive or induced, localized or systemic, and how it correlates with resistance to specific pathogens.

Can CHI-B antibody be used in comparative studies across different plant species?

While the CHI-B antibody is specifically designed for Arabidopsis thaliana (P19171) , it may cross-react with homologous proteins in closely related species depending on sequence conservation. For comparative studies, researchers should perform preliminary validation tests to determine cross-reactivity. If direct cross-reactivity is limited, the antibody can still be valuable as a reference point for developing or selecting antibodies for other plant species, enabling broader comparative studies of chitinase function across the plant kingdom.

What are the optimal conditions for using CHI-B antibody in Western blotting?

While specific optimization parameters for CHI-B antibody aren't provided in the search results, general best practices for plant protein antibodies include:

• Sample preparation:

  • Efficient extraction buffer (typically containing detergents and protease inhibitors)

  • Appropriate protein quantification method

  • Loading 20-50 μg of total protein per lane

• Transfer conditions:

  • PVDF membrane typically offers better protein retention for plant proteins

  • Semi-dry or wet transfer methods are both applicable

• Blocking and antibody incubation:

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

  • Primary antibody dilution should be optimized (typically starting at 1:1000)

  • Overnight incubation at 4°C often yields best results

  • Secondary antibody selection should match the host species of the primary antibody

These parameters should be empirically optimized for CHI-B detection in specific experimental contexts.

What protocols can be used for immunoprecipitation with CHI-B antibody?

For immunoprecipitation of CHI-B from plant tissues:

• Prepare plant lysate in a non-denaturing buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • Protease inhibitor cocktail

• Pre-clear lysate with protein A/G beads

• Incubate pre-cleared lysate with CHI-B antibody (2-5 μg per 1 mg of total protein) overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads thoroughly (typically 4-5 times) with buffer containing reduced detergent

• Elute bound proteins by boiling in SDS-PAGE sample buffer or by using a specific elution buffer

• Analyze by SDS-PAGE followed by Western blotting or mass spectrometry

This protocol may require optimization based on specific research needs and the binding characteristics of the CHI-B antibody.

How should CHI-B antibody be stored and handled for maximum effectiveness?

Based on standard antibody handling practices:

• Store lyophilized antibody at -20°C until reconstitution

• Upon reconstitution, aliquot to avoid repeated freeze-thaw cycles

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

• For short-term use (within 1-2 weeks), store at 4°C

• Avoid exposure to light for fluorescently conjugated antibodies

• Follow manufacturer's recommendations for specific storage buffer compositions

• The typical 2ml/0.1ml size offered by the supplier suggests available concentrated and working dilution formats

What are common issues when using CHI-B antibody and how can they be resolved?

How can researchers validate the specificity of CHI-B antibody?

Validation strategies for CHI-B antibody should include:

• Peptide competition assay: Pre-incubating the antibody with purified CHI-B protein or the immunizing peptide should abolish specific signal

• Genetic controls: Testing samples from CHI-B knockout/knockdown plants versus wild-type

• Overexpression validation: Using samples from plants overexpressing CHI-B to confirm increased signal

• Immunoprecipitation followed by mass spectrometry: To confirm that the antibody is pulling down the correct protein

• Western blot analysis: Confirming that the detected protein is of the expected molecular weight

Similar approaches were used to validate the specificity of monoclonal antibodies against midge hemoglobin component III (Chi t I), where synthetic peptides were used to map binding epitopes .

What factors affect CHI-B protein expression that might impact antibody detection?

Several factors can influence CHI-B expression levels in plants:

• Pathogen exposure: Chitinases are often pathogenesis-related proteins induced during fungal infection

• Developmental stage: Expression may vary throughout plant development

• Tissue specificity: Different plant tissues may express varying levels of CHI-B

• Environmental stressors: Drought, salinity, and temperature extremes may alter expression

• Circadian regulation: Expression levels might fluctuate throughout the day

• Hormone treatments: Plant hormones like ethylene, jasmonic acid, and salicylic acid may induce expression

Researchers should consider these factors when designing experiments and interpreting results from CHI-B antibody-based assays.

How is CHI-B antibody being used in cutting-edge plant immunity research?

Current research applications include:

• Studying the spatial and temporal dynamics of CHI-B induction during pathogen infection using immunolocalization

• Investigating CHI-B protein-protein interactions through co-immunoprecipitation followed by mass spectrometry

• Examining post-translational modifications of CHI-B during immune responses

• Exploring the role of CHI-B in non-host resistance mechanisms

• Studying the coordination between multiple chitinases in plant defense systems, similar to the synergistic effects observed in bacterial chitinase systems where combinations of chitinases showed approximately 2.0-fold increase in hydrolytic activity compared to individual enzymes

What synergistic effects might CHI-B have with other chitinases?

Drawing parallels from bacterial chitinase research, we can hypothesize that plant CHI-B might work synergistically with other chitinases. In Alteromonas sp. strain O-7, the proper combination of four chitinases (ChiA, ChiB, ChiC, and ChiD) resulted in approximately 2.0-fold increase in hydrolytic activity against powdered chitin . This suggests that different chitinases may have complementary roles, with some being more effective against certain substrate forms or during different stages of chitin degradation. Investigation of similar synergistic effects among plant chitinases, including CHI-B, represents an important research direction.

How can advanced imaging techniques enhance CHI-B antibody applications?

Emerging imaging applications include:

• Super-resolution microscopy to visualize subcellular localization of CHI-B with nanometer precision

• Live-cell imaging using fluorescently-tagged antibody fragments to track CHI-B dynamics

• Correlative light and electron microscopy (CLEM) to combine functional and structural information about CHI-B localization

• Proximity ligation assays to detect protein-protein interactions involving CHI-B in situ

• Expansion microscopy to achieve improved resolution of CHI-B distribution in plant tissues

These advanced techniques can provide unprecedented insights into the spatial and temporal aspects of CHI-B function during plant immune responses.