CBP60D Antibody

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Description

CBP60 Protein Family Overview

The CBP60 (CALMODULIN-BINDING PROTEIN 60) family in Arabidopsis includes CBP60a–g and SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1). These proteins are transcription factors with roles in immunity, stress responses, and growth regulation .

ProteinFunctionRegulatory Role
CBP60gImmune gene activation (e.g., SID2, EDS1)Positive regulator of pathogen-induced SA biosynthesis
SARD1Redundant with CBP60g in immune signalingRequired for SA accumulation and defense gene expression
CBP60bBasal defense and NLR-mediated autoimmunityDual role: activates immunity genes but is monitored by NLRs
CBP60aImmune suppressionNegative regulator of immunity

Antibody Validation Challenges

While antibodies against plant proteins like CBP60g or SARD1 are critical for research, commercial antibodies often lack specificity. A 2023 study evaluated 614 antibodies targeting human proteins and found that ~50% failed in at least one application (e.g., Western blot, immunofluorescence). Recombinant antibodies performed better than monoclonal/polyclonal variants .

Key Findings on Antibody Performance:

  • Western Blot (WB): 55/65 proteins had at least one functional antibody.

  • Immunoprecipitation (IP): Success rate varied widely depending on target solubility.

  • Immunofluorescence (IF): Required high-affinity antibodies for reliable detection .

Evolution of Antibody Responses

Studies on SARS-CoV-2 antibodies highlight principles relevant to plant immunity:

  • Neutralizing antibodies evolve over time, gaining potency and breadth against viral variants .

  • Memory B cells produce antibodies with improved binding (e.g., EC50 reduced by ~40% over 6 months) .

  • Techniques like LIBRA-seq enable isolation of rare, broadly reactive antibodies .

Research Gaps and Recommendations

  • No CBP60D protein or antibody has been documented in peer-reviewed studies. The nomenclature may refer to a misannotated or hypothetical member of the CBP60 family.

  • Validation protocols for plant antibodies should follow standards like those in human studies , including knockout controls and multi-application testing.

For further clarity, researchers are advised to:

  1. Verify the nomenclature of "CBP60D" against genomic databases (e.g., TAIR, UniProt).

  2. Explore antibodies for well-characterized CBP60 members (e.g., Anti-CBP60g [Agrisera, AS12 1852]) as alternatives.

  3. Consult structural predictions in CBP60-DB to identify conserved domains for antibody design .

Product Specs

Buffer
**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
CBP60D antibody; At4g25800 antibody; F14M19.80Calmodulin-binding protein 60 D antibody
Target Names
CBP60D
Uniprot No.

Target Background

Function
CBP60D Antibody is a transcription activator that binds DNA in a sequence-specific manner, likely 5'-GAAATTTTGG-3', to promote the expression of target genes.
Database Links

KEGG: ath:AT4G25800

STRING: 3702.AT4G25800.1

UniGene: At.28145

Protein Families
Plant ACBP60 protein family
Subcellular Location
Nucleus.
Tissue Specificity
Expressed in leaves, stems, flowers and root.

Q&A

What is CBP60D and how does it relate to other CBP60 family proteins?

CBP60D belongs to the Calmodulin-Binding Protein 60 (CBP60) family, which includes members like CBP60g and SARD1 that play critical roles in plant immunity pathways. CBP60g and SARD1 have been characterized as partially redundant proteins required for activation of salicylic acid (SA) production and other defense responses . While specific literature on CBP60D is limited in the provided search results, it is likely functionally related to these family members, potentially with temporal expression patterns similar to CBP60g, which shows stronger effects early in defense responses, while SARD1 demonstrates stronger effects later .

What are the typical applications for CBP60D antibodies in research?

CBP60D antibodies are valuable tools for investigating plant immune responses. Based on research with similar antibodies, key applications include:

  • Western blot analysis to detect protein expression and quantify levels

  • Immunoprecipitation to study protein-protein interactions

  • Immunofluorescence to determine subcellular localization

  • Chromatin immunoprecipitation (ChIP) to analyze DNA-binding activity

When designing experiments with CBP60D antibodies, researchers should include appropriate controls to validate specificity, similar to those used for antibodies like Carboxypeptidase B2/CPB2, which are validated through direct ELISAs and Western blots with specificity testing against recombinant proteins .

How should I validate a commercial CBP60D antibody before experimental use?

Thorough validation is essential before using any antibody in critical experiments:

  • Perform Western blot analysis on plant tissue samples (both wild-type and cbp60d mutant if available)

  • Conduct specificity testing against recombinant CBP60D and related family proteins (especially CBP60g and SARD1) to assess cross-reactivity

  • Verify consistent band detection at the expected molecular weight (typically between 45-60 kDa for CBP60 family proteins)

  • Test under both reducing and non-reducing conditions as demonstrated in protocols for similar antibodies

  • Include positive controls (tissues known to express CBP60D) and negative controls (tissues with minimal expression)

What are the optimal conditions for using CBP60D antibodies in Western blot analysis?

Based on protocols used for similar antibodies, the following methodology is recommended:

  • Sample preparation:

    • Extract proteins using a buffer containing protease inhibitors

    • For plant tissues, use 100-200 mg tissue per mL of extraction buffer

    • Denature samples at 95°C for 5 minutes in loading buffer

  • Gel electrophoresis and transfer:

    • Use 10-12% SDS-PAGE gels for optimal resolution

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

    • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Antibody incubation:

    • Dilute primary antibody to 1-2 μg/mL in blocking buffer

    • Incubate overnight at 4°C with gentle rocking

    • Use HRP-conjugated secondary antibody at 1:2000-1:5000 dilution

  • Detection:

    • Use enhanced chemiluminescence (ECL) substrate

    • Expect bands at approximately 45-55 kDa, based on similar proteins

How can I design experiments to study CBP60D function during plant immune responses?

When investigating CBP60D's role in plant immunity, consider the following experimental design:

  • Temporal expression analysis:

    • Monitor CBP60D expression at different timepoints after pathogen challenge (0, 3, 6, 12, 24, 48 hours)

    • Compare expression patterns with CBP60g and SARD1, noting that family members may show different temporal dynamics

  • Loss-of-function studies:

    • Generate or obtain cbp60d mutant plants

    • Create double or triple mutants with cbp60g and sard1 to assess functional redundancy

    • Challenge with pathogens and measure defense outputs (SA production, PR gene expression)

  • Protein interaction studies:

    • Use co-immunoprecipitation with CBP60D antibodies to identify interaction partners

    • Confirm interactions with reciprocal IPs and controls

    • Investigate calcium-dependent interactions, as CBP60 proteins are calmodulin-binding proteins

  • Transcriptional regulation:

    • Perform ChIP using CBP60D antibodies to identify DNA binding sites

    • Compare with known CBP60g and SARD1 targets to identify unique and shared targets

What controls should be included when using CBP60D antibodies in immunoassays?

Rigorous controls are essential for obtaining reliable results with CBP60D antibodies:

  • Specificity controls:

    • Include samples from cbp60d knockout/knockdown plants

    • Test for cross-reactivity with recombinant CBP60 family proteins

    • Pre-absorb antibody with recombinant CBP60D to confirm signal specificity

  • Loading controls:

    • Use antibodies against constitutively expressed proteins (actin, tubulin, GAPDH)

    • Apply equal protein loading verified by total protein stains

  • Technical controls:

    • Include secondary antibody-only controls to assess background

    • Use positive control samples with known CBP60D expression

    • Include both unstressed and stressed plant samples, as CBP60 family proteins are typically stress-responsive

How can I use CBP60D antibodies to study protein-protein interactions in plant immunity?

Investigating CBP60D interactions requires careful experimental design:

  • Co-immunoprecipitation (Co-IP):

    • Prepare protein extracts under non-denaturing conditions

    • Use CBP60D antibody coupled to protein A/G beads

    • Perform IP under different Ca²⁺ concentrations to identify calcium-dependent interactions

    • Analyze precipitated proteins by mass spectrometry or Western blot

  • Proximity-dependent labeling:

    • Generate fusion proteins (CBP60D-BioID or CBP60D-TurboID)

    • Express in plant cells and activate labeling during immune response

    • Purify biotinylated proteins and identify by mass spectrometry

    • Confirm interactions using CBP60D antibodies in reverse Co-IP experiments

  • Bimolecular fluorescence complementation (BiFC):

    • Create CBP60D-YFP fragment fusions

    • Co-express with candidate interactors fused to complementary YFP fragments

    • Visualize interactions using confocal microscopy

    • Validate interactions biochemically using CBP60D antibodies

What approaches can be used to understand CBP60D-specific binding targets versus those shared with other family members?

Distinguishing CBP60D-specific functions from those shared with family members requires specialized approaches:

  • Comparative ChIP-seq analysis:

    • Perform ChIP-seq using antibodies against CBP60D, CBP60g, and SARD1

    • Identify unique and overlapping binding sites

    • Validate specific targets with ChIP-qPCR using CBP60D antibodies

  • Protein binding microarrays:

    • Express recombinant CBP60D protein

    • Probe DNA microarrays to identify binding motifs

    • Compare with known binding preferences of CBP60g and SARD1

    • Validate in vivo using CBP60D antibodies in ChIP experiments

  • Selective complementation:

    • Create domain-swapped chimeric proteins between CBP60D and other family members

    • Express in appropriate mutant backgrounds

    • Use CBP60D antibodies to confirm expression and localization

    • Assess functional complementation through defense phenotyping

  • Temporal dynamics analysis:

    • Compare expression timing of CBP60D with CBP60g and SARD1 following pathogen challenge

    • Based on family member patterns, CBP60D may show distinct temporal dynamics similar to how CBP60g acts earlier while SARD1 functions later in defense responses

How can customized binding specificity approaches be applied to generate CBP60D-specific antibodies?

Based on advanced antibody engineering techniques, researchers can enhance CBP60D antibody specificity:

  • Phage display selection with negative selection steps:

    • Implement a biophysics-informed model that associates distinct binding modes with specific ligands

    • Use phage display with selection against CBP60D while performing counter-selection against related family members

    • Sequence selected antibodies and identify specificity-determining residues

    • Validate specificity through direct binding assays against all family members

  • Computational antibody engineering:

    • Apply computational models to predict and design antibody variants with enhanced specificity

    • Generate antibody sequences with customized specificity profiles that target unique epitopes on CBP60D

    • Optimize CDR3 regions, as these are critical determinants of specificity

    • Validate experimentally using binding assays against CBP60D and related proteins

  • Epitope mapping and targeted antibody generation:

    • Identify unique epitopes on CBP60D that are not conserved in other family members

    • Design immunization strategies targeting these unique regions

    • Screen for antibodies with high specificity using both positive and negative selection criteria

    • Characterize binding modes to ensure specificity for intended targets

What might cause inconsistent results when using CBP60D antibodies?

Several factors can contribute to experimental variability:

  • Antibody stability issues:

    • Repeated freeze-thaw cycles can reduce antibody activity

    • Store antibodies in small aliquots at -20°C to -70°C for up to 6 months

    • For short-term storage (up to 1 month), keep at 2-8°C under sterile conditions

  • Sample preparation variables:

    • Inconsistent extraction methods

    • Degradation due to insufficient protease inhibitors

    • Incomplete denaturation for Western blot samples

    • Variable protein loading between experiments

  • Technical factors:

    • Variation in transfer efficiency during Western blotting

    • Inconsistent blocking procedures leading to different background levels

    • Variable incubation times and temperatures

    • Detection system sensitivity fluctuations

  • Biological variables:

    • Plant growth conditions affecting CBP60D expression

    • Developmental stage differences between experiments

    • Stress conditions inadvertently triggering immune responses

    • Circadian regulation of defense-related proteins

How can I quantitatively analyze Western blot data for CBP60D expression studies?

For reliable quantitative analysis:

  • Image acquisition:

    • Use a digital imaging system with a wide dynamic range

    • Avoid saturating the signal (check histogram during capture)

    • Capture multiple exposures to ensure linearity

  • Normalization approach:

    • Normalize to loading controls (housekeeping proteins)

    • Consider total protein normalization using stain-free technology

    • Include calibration standards when possible

  • Analysis methodology:

    • Use software that can accurately quantify band intensity

    • Subtract local background for each lane

    • Generate standard curves when absolute quantification is needed

    • Calculate relative expression compared to control samples

  • Statistical analysis:

    • Perform experiments with at least three biological replicates

    • Apply appropriate statistical tests (t-test, ANOVA)

    • Report both mean values and measures of variance

    • Consider non-parametric tests if data doesn't meet normality assumptions

How can I address potential cross-reactivity issues with CBP60D antibodies?

When cross-reactivity is suspected:

  • Experimental verification:

    • Test antibody against recombinant CBP60 family proteins

    • Perform Western blots on samples from cbp60d/cbp60g/sard1 single, double, and triple mutants

    • Use peptide competition assays with specific epitope peptides

    • Consider epitope mapping to identify cross-reactive regions

  • Analytical approaches:

    • Compare observed banding patterns with predicted molecular weights

    • Use mass spectrometry to identify proteins in immunoprecipitated samples

    • Perform immunodepletion experiments

  • Alternative methods:

    • Generate epitope-tagged CBP60D for expression studies if antibody specificity cannot be resolved

    • Use transcript analysis (qRT-PCR) to complement protein data

    • Apply CRISPR-based labeling approaches for visualization studies

What are the optimal storage and handling conditions for maintaining CBP60D antibody quality?

To maximize antibody stability and performance:

  • Storage recommendations:

    • Store lyophilized antibodies at -20°C to -70°C for up to 12 months

    • After reconstitution, store at 2-8°C for up to 1 month or at -20°C to -70°C for up to 6 months

    • Avoid repeated freeze-thaw cycles by preparing small working aliquots

  • Reconstitution guidelines:

    • Use sterile techniques when reconstituting lyophilized antibodies

    • Allow vial to reach room temperature before opening

    • Reconstitute in appropriate buffer (usually PBS or manufacturer's recommended buffer)

    • Gentle mixing rather than vortexing to avoid denaturation

  • Working solution preparation:

    • Prepare fresh dilutions on the day of use when possible

    • Use high-quality, filtered buffers

    • Include carrier proteins (BSA, 0.1-0.5%) for dilute solutions

    • Consider adding preservatives for solutions stored more than 24 hours

How can I assess and prevent antibody aggregation and fragmentation?

Antibody quality can be maintained by monitoring and preventing structural changes:

  • Assessment methods:

    • Size exclusion chromatography (SEC) to detect aggregates and fragments

    • SDS-PAGE under non-reducing conditions to identify disulfide-mediated aggregation

    • Dynamic light scattering (DLS) to measure particle size distribution

    • SEC-UV analysis can quantify monomer content, aggregates, and fragments

  • Prevention strategies:

    • Avoid exposure to extreme pH and temperature

    • Minimize exposure to air/liquid interfaces (reduce vortexing/bubbling)

    • Add stabilizers like trehalose or glycerol to storage buffers

    • Filter sterilize antibody solutions to remove nucleation sites for aggregation

  • Monitoring during experiments:

    • Include gel filtration standards as system suitability tests before SEC analysis

    • Monitor changes in retention time or peak shape as indicators of quality issues

    • Implement regular quality control testing of stored antibodies

Antibody StateTypical CompositionDetection Method
Unstressed Antibody92.4% monomer, 7.5% fragments, 0.1% aggregatesSEC-UV analysis
Heat-Stressed Antibody (60°C, 6h)66.3% monomer, 7.4% fragments, 26.4% aggregatesSEC-UV analysis

Table 1: Example composition of unstressed and stressed antibody samples based on SEC-UV analysis

What quality control tests should be performed on CBP60D antibodies before use in critical experiments?

Comprehensive quality control ensures reliable experimental results:

  • Purity assessment:

    • SDS-PAGE analysis under reducing and non-reducing conditions

    • SEC-UV analysis to quantify monomers, aggregates, and fragments

    • Capillary electrophoresis to detect charge variants

  • Functional validation:

    • ELISA binding assays against recombinant CBP60D

    • Western blot using positive control samples

    • Specificity testing against related family members (CBP60g, SARD1)

  • Stability indicators:

    • Test antibody performance after storage at recommended conditions

    • Implement accelerated stability testing if developing custom antibodies

    • Monitor changes in SEC profiles as indicators of physical stability

  • Lot-to-lot consistency:

    • Compare new lots with previously validated lots

    • Establish acceptance criteria for critical quality attributes

    • Maintain reference standards for long-term projects

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