At4g31790 Antibody

What is At4g31790 and why is it significant in plant research?

At4g31790 is a gene identifier in Arabidopsis thaliana that encodes a protein involved in diphthamide biosynthesis pathway. This protein plays a crucial role in post-translational modification of elongation factor 2 (eEF2), which is essential for protein synthesis. The significance of At4g31790 lies in its conservation across eukaryotes and its involvement in fundamental cellular processes. Research suggests this gene may be related to DPH family proteins, particularly DPH1, which is necessary for diphthamide formation in plants .

How are antibodies against plant proteins like At4g31790 typically generated?

Antibodies against plant proteins are typically generated through several established approaches:

• Recombinant protein expression: The coding sequence of At4g31790 is cloned into an expression vector, expressed in bacterial, insect, or yeast systems, purified, and used for immunization.

• Synthetic peptide approach: Short, unique peptide sequences from the At4g31790 protein are synthesized, conjugated to carrier proteins like KLH (keyhole limpet hemocyanin), and used for immunization.

• Immunization protocol: Animals (typically rabbits for polyclonal or mice for monoclonal antibodies) are immunized with the antigen following a prime-boost schedule over 2-3 months.

• Antibody validation: The resulting antibodies must be validated through various techniques including Western blotting, immunoprecipitation, and immunolocalization in both wild-type plants and mutant lines where At4g31790 is absent or modified.

What are the common applications of At4g31790 antibodies in plant research?

At4g31790 antibodies serve multiple critical functions in plant molecular biology research:

• Protein detection and quantification via Western blotting

• Protein localization through immunofluorescence microscopy

• Protein-protein interaction studies via co-immunoprecipitation

• Chromatin immunoprecipitation (ChIP) if the protein has DNA-binding properties

• Functional studies comparing wild-type and mutant plants

• Developmental studies examining protein expression across different tissues and growth stages

Similar to antibodies used to detect diphthamide-modified eEF2 and global eEF2 protein in Arabidopsis, At4g31790 antibodies can be used to investigate specific protein modifications and interactions in different genetic backgrounds .

How should I design experiments to validate At4g31790 antibody specificity?

A robust validation strategy includes:

• Western blot analysis: Compare protein detection in wild-type plants versus knockout/knockdown lines of At4g31790. A specific antibody will show reduced or absent signal in mutant lines.

• Preabsorption testing: Pre-incubate the antibody with purified target protein before immunodetection. Signal reduction confirms specificity.

• Cross-reactivity assessment: Test the antibody against related proteins to ensure it doesn't recognize other family members.

• Multiple antibody comparison: When possible, use antibodies targeting different epitopes of the same protein and compare results.

• Heterologous expression system: Detect the protein in systems where At4g31790 is exogenously expressed versus control systems.

This comprehensive approach, similar to validation methods used for diphthamide-specific antibodies in Arabidopsis , ensures reliable experimental outcomes.

What controls should be included in experiments using At4g31790 antibodies?

Proper implementation of these controls helps distinguish genuine signals from artifacts, similar to approaches used in studies of other plant proteins .

How can At4g31790 antibodies be used to study protein-protein interactions in diphthamide biosynthesis pathways?

At4g31790 antibodies can be powerful tools for investigating protein-protein interactions within diphthamide biosynthesis pathways through several sophisticated techniques:

• Co-immunoprecipitation (Co-IP): At4g31790 antibodies can precipitate the target protein along with its interaction partners from plant cell lysates. These complexes can then be analyzed by mass spectrometry to identify novel interactors or confirm predicted interactions. This approach would be particularly useful for investigating potential interactions between At4g31790 and other DPH family proteins.

• Proximity-dependent biotin identification (BioID): By fusing At4g31790 to a biotin ligase and using the antibody for validation, researchers can identify proximal proteins in living cells.

• Förster resonance energy transfer (FRET): When combined with fluorescently tagged proteins, At4g31790 antibodies can be used to validate FRET experiments examining protein interactions in vivo.

• Yeast two-hybrid validation: At4g31790 antibodies can confirm interactions identified through yeast two-hybrid screens by verifying protein expression and interaction in plant tissues.

These methods could help elucidate whether At4g31790 forms heterodimers with other proteins, similar to the AtDPH1-AtDPH2 heterodimer described in research on diphthamide formation .

What advanced imaging techniques can be combined with At4g31790 antibodies for subcellular localization studies?

Sophisticated imaging approaches for At4g31790 localization include:

• Super-resolution microscopy: Techniques such as Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), and Photoactivated Localization Microscopy (PALM) can provide resolution beyond the diffraction limit (approximately 200 nm), revealing precise localization patterns.

• Correlative Light and Electron Microscopy (CLEM): This technique combines immunofluorescence with electron microscopy to correlate protein localization with ultrastructural features.

• Live-cell imaging with split fluorescent proteins: By fusing complementary fragments of fluorescent proteins to At4g31790 and potential interacting partners, researchers can visualize interactions in real-time when the antibody is used for validation.

• Expansion microscopy: This technique physically expands samples, allowing conventional microscopes to achieve super-resolution imaging of immunolabeled structures.

• Multi-spectral imaging: Using antibodies labeled with different fluorophores can enable simultaneous detection of At4g31790 alongside other proteins of interest.

These advanced techniques could reveal whether At4g31790 localizes to specific cellular compartments, similar to studies showing cytosolic localization of other DPH family proteins in plants .

How can I troubleshoot false negative results when using At4g31790 antibodies?

When facing absence of signal with At4g31790 antibodies, consider these methodological solutions:

• Protein extraction optimization:

  • Try different extraction buffers (varying detergents, salt concentrations)

  • Include protease inhibitors to prevent degradation

  • Test different tissue disruption methods (grinding, sonication)

  • Consider native versus denaturing conditions

• Epitope accessibility issues:

  • If using formaldehyde fixation, test antigen retrieval methods

  • For Western blots, ensure sufficient denaturation of samples

  • Consider membrane type (PVDF vs. nitrocellulose) and transfer conditions

  • Test various blocking reagents (BSA, milk, commercial blockers)

• Antibody optimization:

  • Titrate antibody concentration

  • Extend primary antibody incubation time or temperature

  • Try different detection systems (chemiluminescence, fluorescence)

  • Consider signal amplification methods (TSA, polymeric detection)

• Expression level considerations:

  • Concentrate protein samples if target expression is low

  • Use tissues/conditions with highest expected expression

  • Consider enrichment via immunoprecipitation before detection

These approaches have been successful in optimizing detection of low-abundance plant proteins like those involved in diphthamide modification pathways .

How should I interpret conflicting data between At4g31790 antibody results and transcriptomic data?

Discrepancies between protein and transcript levels are common in biological systems and can reveal important regulatory mechanisms. When facing conflicting data:

• Validate both datasets independently:

  • Confirm antibody specificity with additional controls

  • Verify transcriptomic data with RT-qPCR

  • Use multiple antibodies targeting different epitopes if possible

• Consider post-transcriptional regulation:

  • Examine microRNA targeting of At4g31790 mRNA

  • Investigate mRNA stability factors

  • Assess translation efficiency

• Evaluate post-translational mechanisms:

  • Measure protein half-life (cycloheximide chase)

  • Check for proteasomal degradation (MG132 treatment)

  • Investigate potential protein modifications affecting antibody binding

• Employ complementary approaches:

  • Use GFP/tag fusions to track protein independently

  • Perform targeted proteomics (SRM/MRM/PRM)

  • Consider absolute quantification methods for both protein and mRNA

This approach acknowledges that transcript and protein abundance correlations are often weak in plants. Similar disparities have been observed in previous studies, where "the direction of the change in the abundance of these affected proteins did not necessarily correlate with the change of abundance observed in the transcriptomic data, as seen numerous times before in other reported Plasmodial perturbations" .

What statistical approaches are appropriate for analyzing quantitative data generated with At4g31790 antibodies?

These statistical approaches can be implemented using software packages like R (with packages such as data.table ), providing rigorous analysis of At4g31790 expression data.

How can I develop a quantitative ELISA for At4g31790 protein detection?

Developing a quantitative ELISA for At4g31790 requires systematic optimization:

• Antibody pair selection:

  • Test different capture and detection antibody combinations (monoclonal/polyclonal)

  • Ensure antibodies recognize distinct, non-overlapping epitopes

  • Validate specificity using recombinant protein and plant extracts

• Protocol optimization:

  • Determine optimal coating buffer and concentration for capture antibody

  • Optimize blocking conditions to minimize background

  • Establish ideal sample dilution ranges

  • Develop appropriate standard curves using recombinant protein

• Validation parameters:

  • Limit of detection (LOD) and limit of quantification (LOQ)

  • Intra- and inter-assay coefficients of variation

  • Spike recovery and parallelism tests

  • Cross-reactivity assessment with related proteins

• Data analysis approach:

  • Implement appropriate curve fitting (4-parameter logistic regression)

  • Establish quality control criteria for assay acceptance

  • Develop normalization strategies for sample comparison

The methodology can draw inspiration from antibody-based quantitative assays for other proteins, similar to the approach described for measuring EC50 values of antigen binding, where cadonilimab showed EC50 values of 0.051 nM and 0.079 nM for PD1-mFc and CTLA4-mFc antigens respectively in ELISA assays .

What emerging technologies might enhance the utility of At4g31790 antibodies in plant research?

Several cutting-edge technologies can significantly extend At4g31790 antibody applications:

• Single-cell proteomics:

  • Combining antibodies with mass cytometry (CyTOF) for single-cell protein quantification

  • Microfluidic antibody-based sorting of specific cell populations

  • Single-cell Western blotting for cell-to-cell protein variation analysis

• In vivo antibody applications:

  • Cell-penetrating antibody derivatives for live-cell imaging

  • Nanobody development for improved tissue penetration

  • Optogenetic antibody systems for temporally controlled inhibition

• High-throughput screening platforms:

  • Antibody arrays for parallel protein quantification

  • Automated immunoprecipitation-mass spectrometry workflows

  • Microfluidic immunoassays for rapid analysis

• Computational approaches:

  • Machine learning algorithms for improved Western blot quantification

  • Integrative multi-omics analysis incorporating antibody-based data

  • Automated image analysis for complex colocalization patterns

• Antibody engineering:

  • CRISPR-based epitope tagging for improved antibody recognition

  • Recombinant antibody fragments with enhanced specificity

  • Bispecific antibodies for detecting protein complexes

These advances could transform our understanding of At4g31790 biology, similar to how tetravalent bispecific antibody designs have enhanced target binding through increased avidity in high-density co-expression contexts .