At2g21725 Antibody

What is the AT2G07725 antibody and what protein does it target?

The AT2G07725 antibody is a research tool designed to specifically recognize and bind to the 60S ribosomal protein L5 located in plant mitochondria. This antibody targets a critical component of the mitochondrial ribosomal machinery involved in protein synthesis within the organelle. The antibody was generated using synthetic peptides that share 100% sequence homology with the RPL5 protein sequence (ATMG00210), making it highly specific for research applications examining mitochondrial ribosomal structure and function in plants .

What plant species show cross-reactivity with this antibody?

The AT2G07725 antibody demonstrates extensive cross-reactivity across multiple plant species due to the high conservation of the ribosomal L5 protein sequence. Based on empirical testing, the antibody effectively recognizes target proteins in the following species:

This broad cross-reactivity makes the antibody a versatile tool for comparative studies across different plant taxa .

What are the optimal storage conditions for maximizing antibody stability and performance?

For optimal antibody performance and longevity, the AT2G07725 antibody should be stored according to these evidence-based protocols:

• Upon receipt, immediately store the lyophilized antibody in a manual defrost freezer

• Minimize freeze-thaw cycles as they significantly degrade antibody function and specificity

• When thawing for use, maintain at 4°C during experimental procedures

• For long-term storage, aliquot reconstituted antibody to minimize freeze-thaw cycles

• Document storage conditions and freeze-thaw events to correlate with experimental performance

Following these storage protocols has been demonstrated to preserve antibody activity and reduce batch-to-batch variation in experimental outcomes .

How can I optimize antibody dilution for different experimental applications?

Establishing optimal antibody dilution is critical for achieving robust and reproducible results. The following methodological approach is recommended:

• Perform a preliminary dilution series (1:100, 1:500, 1:1000, 1:5000) for your specific application

• Include both positive and negative controls to establish signal-to-noise ratio

• For Western blotting: Begin with 1:1000 dilution in 5% BSA or milk blocker

• For immunohistochemistry: Start with 1:200 dilution and adjust based on signal intensity

• For ELISA applications: Test dilutions between 1:500 and 1:2000

Optimized dilution factors should be determined empirically for each experimental technique, cell/tissue type, and detection system to ensure balance between specific signal and background noise .

How can AT2G07725 antibody be utilized in multi-parameter immunofluorescence studies?

For complex multi-parameter immunofluorescence experiments examining plant mitochondrial complexes, researchers should employ this methodological workflow:

• Sample preparation optimization:

  • Use fresh tissue samples or properly fixed specimens (4% paraformaldehyde for 15-20 minutes)

  • Perform antigen retrieval using citrate buffer (pH 6.0) heating method if necessary

  • Include permeabilization step with 0.1% Triton X-100 to facilitate antibody access to mitochondrial targets

• Antibody multiplexing strategy:

  • Utilize AT2G07725 as primary antibody in combination with antibodies against other mitochondrial components

  • Select complementary fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Implement sequential staining for antibodies derived from the same species

  • Include DAPI nuclear counterstain and MitoTracker for organelle localization

• Control implementation:

  • Run parallel slides with isotype controls at matching concentrations

  • Include single-antibody controls to assess bleed-through in multiplexed channels

  • Prepare absorption controls using immunizing peptide

This methodological approach enables visualization of L5 protein in context with other mitochondrial components, providing insights into ribosomal assembly and function .

What strategies can improve antibody detection of low-abundance mitochondrial proteins?

When investigating low-abundance mitochondrial ribosomal proteins, researchers can employ these advanced techniques to enhance detection sensitivity:

• Sample enrichment:

  • Perform mitochondrial isolation using differential centrifugation

  • Further fractionate mitochondrial lysates to concentrate ribosomal components

  • Implement immunoprecipitation to concentrate target proteins before analysis

• Signal amplification methods:

  • Utilize tyramide signal amplification (TSA) for immunohistochemistry

  • Employ biotin-streptavidin amplification systems

  • Consider implementing quantum dot-conjugated secondary antibodies for enhanced signal stability

• Detection optimization:

  • Use highly sensitive ECL substrates for Western blotting

  • Extend primary antibody incubation time (overnight at 4°C)

  • Implement epitope retrieval techniques specific for mitochondrial proteins

These methodological enhancements have demonstrated up to 10-fold increases in detection sensitivity for low-abundance mitochondrial proteins like L5 .

What methods should be used to validate antibody specificity for ribosomal proteins?

Rigorous validation of antibody specificity is essential for reliable research outcomes. For AT2G07725 antibody, implement this comprehensive validation workflow:

• Western blot analysis:

  • Confirm single band at expected molecular weight (~34 kDa for L5)

  • Test with recombinant protein controls

  • Compare reactivity in wild-type vs. L5 knockdown/knockout models

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Parallel testing of blocked vs. unblocked antibody

  • Evaluate signal reduction to confirm epitope specificity

• Mass spectrometry validation:

  • Immunoprecipitate target protein using AT2G07725 antibody

  • Perform LC-MS/MS analysis of precipitated proteins

  • Confirm presence of target protein and associated complex components

• Orthogonal method correlation:

  • Compare protein detection with alternative antibodies targeting different epitopes

  • Correlate protein levels with mRNA expression data

  • Use GFP-tagged constructs as complementary detection method

How can I assess batch-to-batch consistency for critical experiments?

Maintaining experimental reproducibility across antibody batches requires systematic quality control measures:

• Reference standard creation:

  • Establish a well-characterized positive control sample

  • Create sufficient aliquots for testing multiple antibody batches

  • Document signal intensity under standardized conditions

• Quantitative comparison protocol:

  • Perform side-by-side testing of new and reference antibody batches

  • Use quantitative Western blot or ELISA to determine relative potency

  • Calculate a normalization factor if necessary for data consistency

• Specificity confirmation:

  • Verify that epitope recognition pattern remains consistent

  • Check for any new non-specific bands or cross-reactivity

  • Confirm signal-to-noise ratio meets established thresholds

Implementation of these quality control measures minimizes experimental variability and enhances data reproducibility across extended research projects .

What are common causes of false negative results and their solutions?

False negative results when using AT2G07725 antibody can stem from multiple methodological factors. The following systematic approach addresses common issues:

This structured approach allows researchers to systematically isolate and address technical issues affecting experimental outcomes .

How can I minimize background while maximizing specific signal?

Optimizing signal-to-noise ratio is crucial for generating publication-quality data. Implement these evidence-based strategies:

• Blocking optimization:

  • Compare different blocking agents (BSA, milk, commercial blockers)

  • Titrate blocking agent concentration (1-5%)

  • Extend blocking time to reduce non-specific binding

• Washing protocol refinement:

  • Increase washing buffer volume and duration

  • Add detergents (0.05-0.1% Tween-20) to reduce hydrophobic interactions

  • Implement additional washing steps after secondary antibody incubation

• Antibody preparation:

  • Pre-adsorb antibodies against common cross-reactive components

  • Centrifuge antibody solutions before use to remove aggregates

  • Prepare fresh dilutions for each experiment

• Detection system optimization:

  • Select detection reagents matched to expression level

  • Use enzyme substrates with lower background characteristics

  • Implement shorter substrate development times with monitoring

These methodological refinements significantly enhance signal specificity while minimizing background interference .

How can the AT2G07725 antibody be applied in plant stress response studies?

The AT2G07725 antibody offers valuable research applications for investigating mitochondrial ribosomal responses to environmental stressors:

• Experimental design for stress response studies:

  • Establish baseline L5 protein expression under normal conditions

  • Apply controlled stress treatments (drought, salt, temperature, oxidative stress)

  • Track temporal changes in L5 protein levels, post-translational modifications, and localization

• Multi-omics integration approach:

  • Correlate L5 protein levels with transcriptomic changes in nuclear and mitochondrial genes

  • Analyze mitochondrial translation efficiency under stress conditions

  • Assess impacts on respiratory complex assembly and function

• Comparative analysis across species:

  • Leverage the broad cross-reactivity to examine evolutionary conservation of stress responses

  • Compare L5 dynamics across species with different stress tolerance mechanisms

  • Identify potential regulatory patterns for mitochondrial translation during stress adaptation

This research approach provides insights into how plant mitochondrial translation machinery responds to environmental challenges, with potential applications for crop improvement .

What are the considerations for using AT2G07725 antibody in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with AT2G07725 antibody enables investigation of mitochondrial ribosomal protein interactions. Researchers should implement this methodological framework:

• Sample preparation optimization:

  • Utilize gentle lysis buffers to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to maintain complex integrity

  • Test different detergent types and concentrations to balance solubilization and complex preservation

• Antibody immobilization strategies:

  • Compare direct antibody conjugation vs. protein A/G approaches

  • Optimize antibody-to-bead ratio (typically 5-10 μg antibody per 50 μl bead volume)

  • Determine ideal cross-linking conditions if needed to prevent antibody leaching

• Interaction validation:

  • Implement reciprocal Co-IP with antibodies against suspected interaction partners

  • Include appropriate negative controls (isotype antibodies, irrelevant targets)

  • Perform stringency washes to eliminate non-specific binding

• Analysis considerations:

  • Use mass spectrometry for unbiased identification of interaction partners

  • Validate key interactions with orthogonal methods (proximity ligation, FRET)

  • Assess interaction dynamics under different physiological conditions

This structured Co-IP methodology facilitates reliable characterization of mitochondrial ribosomal complexes and their dynamic regulation .

How might nanobody technology improve mitochondrial ribosomal protein research?

While traditional antibodies like AT2G07725 have been research mainstays, emerging nanobody technology offers potential advantages for mitochondrial research:

• Nanobody advantages for mitochondrial applications:

  • Smaller size (12-15 kDa vs. 150 kDa) enables better penetration into mitochondrial structures

  • Enhanced recognition of conformational epitopes in native protein complexes

  • Greater stability under various experimental conditions

  • Potential for intracellular expression as research tools

• Development considerations:

  • Generation of nanobodies against AT2G07725 target epitopes

  • Validation against traditional antibody performance

  • Optimization for live-cell imaging applications

• Research applications:

  • Super-resolution microscopy of mitochondrial ribosome dynamics

  • Intracellular tracking of ribosomal assembly

  • Targeted modulation of mitochondrial translation

The integration of nanobody technology represents a promising frontier for enhancing mitochondrial ribosomal protein research with improved spatial and temporal resolution .

What computational approaches can enhance antibody design and application?

Advanced computational methods offer opportunities to improve antibody research tools and applications:

• Structure-based antibody engineering:

  • Computational prediction of epitope accessibility in target proteins

  • In silico affinity maturation to enhance binding properties

  • Design of antibodies targeting conformational epitopes in ribosomes

• Application in experimental design:

  • Modeling of antibody binding to complex ribosomal structures

  • Prediction of optimal epitope targeting for specific applications

  • Computational assessment of cross-reactivity with related proteins

• Integration with experimental data:

  • Combining structural modeling with empirical binding data

  • Refinement of antibody properties based on experimental feedback

  • Development of improved variants through iterative computational design

These computational approaches can assist in developing next-generation antibody tools with enhanced specificity, affinity, and application-specific performance characteristics .