CID7 Antibody

What is CID7 protein and why is it significant in plant research?

CID7 (Cap-binding protein Interacting Domain 7) is a protein found in Arabidopsis thaliana that plays important roles in plant cellular processes. The protein has been identified in biochemical studies as part of plant stress response pathways and may function in RNA processing mechanisms. CID7 antibodies are critical research tools that allow scientists to detect, quantify, and isolate this protein from plant tissues, enabling studies of its expression patterns, subcellular localization, and protein-protein interactions.

The CID7 antibody currently available for research is a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana CID7 protein as the immunogen . This antibody has been affinity-purified to enhance specificity and is formulated in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Understanding the properties of this antibody is essential for designing reliable experimental approaches in plant biology research.

What are the validated applications for CID7 antibody?

The CID7 antibody has been validated for several key molecular biology techniques commonly used in plant research. According to available specifications, the primary validated applications include Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These techniques allow researchers to detect and quantify CID7 protein in complex biological samples.

In Western blotting applications, the CID7 antibody enables detection of the target protein in plant tissue extracts following separation by SDS-PAGE and transfer to a membrane. This application is particularly valuable for studying protein expression levels under different experimental conditions or developmental stages. For quantitative analysis, ELISA provides a sensitive method for measuring CID7 protein concentration in plant extracts without the need for protein separation.

While not explicitly validated, researchers may adapt this antibody for additional applications such as immunoprecipitation, immunohistochemistry, or flow cytometry after performing appropriate validation experiments. When using the antibody for non-validated applications, thorough controls must be included to ensure specificity and reliability of results.

How should CID7 antibody be stored and handled to maintain its activity?

Proper storage and handling of antibodies is crucial for maintaining their activity and specificity over time. For CID7 antibody, the manufacturer recommends storage at -20°C or -80°C upon receipt . The antibody should be aliquoted to avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of binding activity.

The storage buffer containing 50% glycerol helps prevent freezing damage, but it remains important to handle the antibody with care during experiments. When working with the antibody, it should be kept on ice or at 4°C and returned to storage promptly after use. The working dilution should be prepared fresh for each experiment to ensure optimal performance.

Documentation of antibody lot numbers, receipt dates, and freeze-thaw cycles is recommended as part of good laboratory practice to track antibody performance over time. If a decrease in antibody performance is observed, this information can help determine whether storage issues might be contributing to the problem.

What controls should be incorporated when working with CID7 antibody?

Proper experimental controls are essential for interpreting results obtained with CID7 antibody. A comprehensive set of controls should include:

• Positive Control: Samples known to express CID7 protein, such as specific Arabidopsis tissues or recombinant CID7 protein. This validates that the detection system is working properly.

• Negative Control: Samples from CID7 knockout plants or tissues known not to express CID7. This helps establish the background signal level.

• Loading Control: Detection of a housekeeping protein (e.g., actin, tubulin) to normalize for variations in sample loading and protein extraction efficiency.

• Primary Antibody Control: Omitting the primary antibody while maintaining all other aspects of the protocol to identify non-specific binding of the secondary antibody.

• Secondary Antibody Control: Using an isotype-matched irrelevant primary antibody to identify non-specific binding.

For quantitative applications such as Western blotting densitometry or ELISA, standard curves should be generated using purified recombinant CID7 protein at known concentrations. This enables accurate quantification of CID7 in experimental samples.

How can researchers optimize Western blot protocols for CID7 detection?

Western blot optimization for CID7 detection requires systematic adjustment of several parameters to achieve optimal signal-to-noise ratio. Based on general principles for plant protein detection:

Systematic optimization of these parameters for the specific CID7 antibody will result in reproducible and specific detection of the target protein.

What approaches can address non-specific binding issues with CID7 antibody?

Non-specific binding is a common challenge when working with polyclonal antibodies in plant systems. If encountering high background or non-specific bands when using CID7 antibody, consider the following strategies:

• Increase Blocking Time/Concentration: Extending blocking time to 2 hours or overnight, or increasing the concentration of blocking agent (e.g., from 3% to 5% BSA) can reduce non-specific binding.

• Adjust Antibody Concentration: Titrate the antibody to find the optimal concentration that provides specific signal with minimal background. This might require testing dilutions ranging from 1:500 to 1:5000.

• Add Competing Proteins: Adding 1-5% of the species in which the secondary antibody was raised (e.g., goat serum if using anti-rabbit secondary raised in goat) to the antibody dilution buffer can reduce non-specific binding.

• Modify Washing Conditions: Increase the number, duration, or stringency of washes by adding more Tween-20 (up to 0.3%) or including low concentrations of SDS (0.01-0.05%).

• Pre-adsorption: If cross-reactivity with specific plant proteins is observed, pre-incubating the antibody with extracts from negative control tissues can reduce non-specific binding.

• Alternative Blocking Agents: If standard blocking agents are ineffective, try alternatives such as fish gelatin, casein, or commercial blocking buffers optimized for plant samples.

These approaches can be tested systematically, changing one variable at a time, to identify the optimal conditions for specific detection of CID7 protein.

How can CID7 antibody be used to study protein-protein interactions?

CID7 antibody can be employed in several techniques to investigate protein-protein interactions in plant systems:

• Co-Immunoprecipitation (Co-IP): CID7 antibody can be used to precipitate CID7 protein along with its interacting partners from plant extracts. The precipitated complex can then be analyzed by mass spectrometry or Western blotting to identify interacting proteins.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity. It combines antibody recognition with DNA amplification to generate fluorescent signals only when two proteins are in close proximity.

• Chromatin Immunoprecipitation (ChIP): If CID7 functions in DNA-protein complexes, ChIP can be used to identify DNA sequences associated with CID7 protein complexes.

• Bimolecular Fluorescence Complementation (BiFC): While not directly using the antibody, BiFC results can be validated with CID7 antibody by confirming expression of fusion proteins.

When designing co-immunoprecipitation experiments, several considerations are important:

• Extraction Conditions: Use gentle lysis buffers to preserve protein-protein interactions.

• Cross-linking: Consider chemical cross-linking to stabilize transient interactions.

• Negative Controls: Include IgG from the same species as the CID7 antibody to control for non-specific binding.

• Validation: Confirm interactions using reciprocal co-IP or alternative methods.

Table 1: Optimization Parameters for Co-IP with CID7 Antibody

What approaches should be used to study post-translational modifications of CID7?

Post-translational modifications (PTMs) of plant proteins, including CID7, can significantly impact their function, localization, and interactions. Studying PTMs of CID7 requires specialized approaches:

• Phosphorylation Analysis:

  • Use phosphatase inhibitors during extraction

  • Employ phospho-specific detection methods like Phos-tag SDS-PAGE

  • Consider phospho-enrichment techniques before mass spectrometry analysis

  • Validate with phospho-specific antibodies if available

• Ubiquitination and SUMOylation Analysis:

  • Include deubiquitinating enzyme inhibitors in extraction buffers

  • Use denaturing conditions to preserve these modifications

  • Consider expressing tagged ubiquitin/SUMO constructs for enrichment

  • Detect size shifts by Western blotting with CID7 antibody

• Glycosylation Analysis:

  • Use enzymatic deglycosylation followed by Western blotting to detect size shifts

  • Lectin affinity chromatography can enrich glycosylated forms

• Mass Spectrometry Approaches:

  • Immunoprecipitate CID7 using the antibody

  • Analyze by LC-MS/MS with appropriate fragmentation methods

  • Include enrichment steps for specific PTMs

  • Compare PTM profiles under different conditions or treatments

When detecting PTMs using Western blotting with CID7 antibody, be aware that the antibody recognition might be affected by certain modifications, especially if they occur within the epitope region. In such cases, alternative detection methods may be necessary.

How can researchers study CID7 localization in plant tissues?

Studying the subcellular localization of CID7 provides valuable insights into its function. The CID7 antibody can be used for immunolocalization studies in plant tissues with the following considerations:

• Tissue Fixation:

  • Use 4% paraformaldehyde in PBS for 30-60 minutes at room temperature

  • For better preservation of antigenicity, try shorter fixation times or milder fixatives

  • Consider comparing multiple fixation methods to determine optimal conditions

• Tissue Permeabilization:

  • For cell wall permeabilization, use 0.1-0.5% Triton X-100 or 0.05-0.1% Tween-20

  • Enzymatic digestion with cellulase/pectinase may improve antibody accessibility

  • Optimize permeabilization to balance antibody accessibility and structural preservation

• Blocking and Antibody Incubation:

  • Block with 3-5% BSA or 5-10% normal serum from the species of the secondary antibody

  • Incubate with CID7 primary antibody at 1:100 to 1:500 dilution overnight at 4°C

  • For secondary antibody, use fluorophore-conjugated anti-rabbit IgG at 1:200 to 1:1000

• Controls for Immunofluorescence:

  • Negative controls: omit primary antibody or use pre-immune serum

  • Competing peptide control: pre-incubate antibody with excess antigen

  • Positive controls: tissues known to express CID7

  • Counter-staining: use organelle markers to confirm subcellular localization

• Advanced Imaging Techniques:

  • Confocal microscopy for improved resolution and 3D localization

  • Super-resolution microscopy for detailed subcellular structure visualization

  • Co-localization analysis with markers for specific organelles or structures

For dynamic studies of CID7 localization under different conditions, consider:

• Comparing tissues at different developmental stages

• Examining localization changes in response to stress conditions

• Using inducible expression systems to track newly synthesized CID7

How can researchers address inconsistent results with CID7 antibody?

Inconsistent results when using CID7 antibody can stem from multiple sources. A systematic troubleshooting approach includes:

• Antibody Storage and Quality:

  • Aliquot antibody upon receipt to minimize freeze-thaw cycles

  • Test different lots if available and maintain consistent lot usage within experiments

  • Consider performing a simple dot blot to assess antibody activity before complex experiments

• Sample Preparation Variables:

  • Ensure consistent protein extraction methods across experiments

  • Standardize protein quantification methods

  • Include protease inhibitors in all buffers to prevent degradation

  • Process all samples within an experiment simultaneously

• Technical Variations:

  • Standardize incubation times and temperatures

  • Use automated systems where possible to reduce handler variability

  • Prepare fresh working solutions for each experiment

  • Consider using transfer and loading controls like Ponceau S staining

• Experimental Design Improvements:

  • Include technical and biological replicates

  • Implement quantitative methods like densitometry with appropriate normalization

  • Use positive and negative controls in every experiment

  • Consider alternative detection methods if results remain inconsistent

• Statistical Analysis:

  • Apply appropriate statistical tests to determine if variations are significant

  • Consider power analysis to determine the number of replicates needed

  • Use quantitative analysis software to minimize subjective interpretation

Maintaining detailed laboratory records is essential for tracking variables that might affect antibody performance. Document all experimental conditions, reagent lot numbers, and environmental factors for comprehensive troubleshooting.

What strategies can be employed for detecting low-abundance CID7 protein?

Detecting low-abundance proteins like CID7 in plant samples often requires enhanced sensitivity approaches:

• Sample Enrichment Techniques:

  • Subcellular fractionation to concentrate CID7 in relevant fractions

  • Immunoprecipitation to concentrate CID7 before detection

  • Protein precipitation methods to concentrate total protein

• Enhanced Detection Methods:

  • Use high-sensitivity chemiluminescent substrates for Western blotting

  • Consider tyramine signal amplification for immunohistochemistry

  • Try biotin-streptavidin systems for signal enhancement

  • Explore fluorescent detection with high-quantum-yield fluorophores

• Instrumentation Considerations:

  • Use highly sensitive imaging systems with cooled CCD cameras

  • Extend exposure times while monitoring background levels

  • Consider photon-counting techniques for maximum sensitivity

• Protocol Modifications:

  • Increase primary antibody concentration or incubation time

  • Reduce washing stringency while monitoring background

  • Use larger sample volumes or load more total protein

• Alternative Detection Formats:

  • Consider direct ELISA instead of Western blotting for quantification

  • Explore proximity ligation assay (PLA) for in situ detection with amplification

  • Consider MS-based targeted proteomics approaches

Table 2: Sensitivity Enhancement Strategies for CID7 Detection

How can CID7 antibody be validated for cross-species applications?

When extending CID7 antibody use to plant species beyond Arabidopsis thaliana, careful validation is essential:

• Sequence Homology Analysis:

  • Perform bioinformatic analysis of CID7 protein sequence conservation across species

  • Identify regions of high conservation, especially in the likely epitope regions

  • Predict potential cross-reactivity based on sequence identity percentages

• Initial Cross-Reactivity Testing:

  • Perform Western blot analysis with protein extracts from target species

  • Look for bands of expected molecular weight based on predicted CID7 homologs

  • Compare band patterns between Arabidopsis (positive control) and target species

• Validation Approaches:

  • Peptide competition assays to confirm specificity

  • Use of knockout/knockdown lines in target species if available

  • Heterologous expression of target species CID7 homolog as positive control

  • Mass spectrometry validation of immunoprecipitated proteins

• Optimization for Cross-Species Use:

  • Adjust antibody concentration for optimal signal-to-noise ratio in new species

  • Modify extraction buffers to account for species-specific matrix effects

  • Consider species-specific blocking agents to reduce background

• Experimental Design Considerations:

  • Include appropriate positive and negative controls from both species

  • Run parallel experiments with Arabidopsis samples for direct comparison

  • Consider using conserved housekeeping proteins as loading controls

The success of cross-species application depends largely on the conservation of epitope regions between species. Polyclonal antibodies like the CID7 antibody may recognize multiple epitopes, potentially increasing the likelihood of cross-reactivity with homologous proteins in related species.

How can CID7 antibody contribute to functional genomics studies in plants?

CID7 antibody represents a valuable tool for functional genomics approaches in plant biology:

• Integration with Genetic Resources:

  • Analysis of CID7 protein expression in mutant lines

  • Correlation of phenotypic data with protein expression levels

  • Validation of gene editing outcomes at the protein level

  • Assessment of compensatory mechanisms in knockout/knockdown lines

• Systems Biology Applications:

  • Protein expression profiling across developmental stages

  • Response profiling under various stress conditions

  • Integration of proteomics data with transcriptomics and metabolomics

  • Network analysis using protein interaction data

• Emerging Technologies Integration:

  • Single-cell proteomics applications using microfluidics

  • Spatial proteomics using multiplexed immunofluorescence

  • High-throughput screening of plant populations

  • CRISPR-based functional genomics validated at protein level

• Translational Applications:

  • Assessment of CID7 function in crop species

  • Evaluation of CID7 as potential biomarker for plant stress responses

  • Development of biosensors based on CID7 interactions

  • Targeted breeding approaches based on functional proteomics

As plant functional genomics continues to evolve, antibody-based protein detection will remain a cornerstone methodology, complementing newer technologies and providing critical validation for genetic and transcriptomic findings.

What considerations apply to developing next-generation antibodies for plant research?

The future of plant antibody research may involve several advancements that could enhance CID7 detection and similar applications:

• Recombinant Antibody Technologies:

  • Development of single-chain variable fragments (scFvs) against plant proteins

  • Phage display selection for higher specificity antibodies

  • Nanobody development for improved tissue penetration

  • Humanized antibodies for reduced background in certain applications

• Epitope-Specific Approaches:

  • Generation of antibodies against specific post-translationally modified forms

  • Development of conformation-specific antibodies for functional states

  • Linear epitope mapping to improve antibody design

  • Structural biology approaches to identify optimal binding sites

• Multispecificity Considerations:

  • Bispecific antibodies targeting CID7 and interacting partners

  • Multiplexed detection systems with orthogonal labels

  • Modular antibody frameworks for customized applications

  • Cross-species consensus epitope targeting

• Technical Improvements:

  • Enhanced stability antibodies for harsh extraction conditions

  • Reduced cross-reactivity in plant systems

  • Improved signal-to-noise ratio through engineering

  • Direct conjugation to novel reporter systems

While commercial development of plant research antibodies may lag behind those for biomedical research, academic and collaborative initiatives can drive innovation in this field, leading to next-generation reagents with enhanced performance characteristics.