At5g50450 Antibody

What is At5g50450 and why would researchers need an antibody against it?

At5g50450 is an Arabidopsis thaliana gene encoding an HCP-like superfamily protein with a MYND-type zinc finger domain. The protein functions in binding and zinc ion binding . Given its potential role in plant stress responses and transcriptional regulation, researchers utilize antibodies against At5g50450 to study its expression patterns, protein-protein interactions, and subcellular localization, particularly in the context of environmental stress responses and developmental processes.

How should researchers validate the specificity of an At5g50450 antibody?

Validation of At5g50450 antibody specificity requires multiple complementary approaches:

• Western blot analysis using:

  • Wild-type Arabidopsis extracts (positive control)

  • At5g50450 knockout/mutant lines (negative control)

  • Recombinant At5g50450 protein (positive control)

• Immunoprecipitation (IP) followed by mass spectrometry to confirm target identity

• Cross-reactivity testing against closely related zinc finger proteins

• Preabsorption test with recombinant At5g50450 protein to neutralize the antibody

For highest confidence, utilize multiple mutant lines (such as RIKEN 16-0079-1, GT16086, GT19034, or GT24069) as negative controls to ensure the antibody does not produce signals in tissues lacking the target protein.

How can At5g50450 antibody be used to study protein localization in plant cells?

For subcellular localization studies:

• Immunofluorescence microscopy:

  • Fix Arabidopsis seedlings or protoplasts with 4% paraformaldehyde

  • Permeabilize cell walls with enzyme solution (cellulase R10 and macerozyme R10)

  • Block with 3% BSA for 1 hour

  • Incubate with At5g50450 primary antibody (1:100 to 1:500 dilution)

  • Apply fluorophore-conjugated secondary antibody

  • Co-stain with nuclear marker (DAPI) and other organelle markers as needed

• Cell fractionation followed by immunoblotting:

  • Separate nuclear and cytoplasmic fractions using differential centrifugation

  • Perform western blotting with At5g50450 antibody on each fraction

  • Include control antibodies for compartment-specific markers

This approach has successfully determined subcellular localization of other plant proteins such as ATG6 and NPR1 , which showed dual localization in both cytoplasm and nucleus.

How can researchers optimize western blot conditions for At5g50450 detection?

For optimal western blot detection of At5g50450:

• Gel percentage: Use 12% SDS-PAGE gels for optimal separation

• Transfer conditions: Transfer to PVDF membrane at 100V for 1 hour in 10% methanol transfer buffer

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

• Antibody dilution: Start with 1:1,000 to 1:10,000 dilution (optimize empirically)

• Incubation: Overnight at 4°C with gentle rocking

• Detection system: Use chemiluminescence for standard detection; consider fluorescent secondary antibodies for quantitative analysis

For low abundance proteins like transcription factors, include proteasome inhibitors (MG132, 40μM) in extraction buffers and consider using signal enhancers during detection .

How can researchers use At5g50450 antibody to study stress responses in Arabidopsis?

To investigate At5g50450's role in stress responses:

• Comparative expression analysis:

  • Treat Arabidopsis plants with various stressors (salt, drought, cold, heat)

  • Harvest tissues at multiple time points

  • Perform western blotting with At5g50450 antibody

  • Quantify protein levels relative to loading controls

• Chromatin immunoprecipitation (ChIP):

  • Cross-link proteins to DNA in stressed and control plants

  • Immunoprecipitate using At5g50450 antibody

  • Analyze bound DNA by qPCR or sequencing

  • Identify stress-responsive genes regulated by At5g50450

This approach could reveal connections between At5g50450 and stress responses, similar to findings showing that some zinc finger proteins respond to salt stress and auxin signaling pathways .

What approaches can overcome cross-reactivity issues with At5g50450 antibody?

When facing cross-reactivity challenges:

• Epitope selection refinement:

  • Target unique regions of At5g50450 that differ from other MYND-type zinc finger proteins

  • Avoid conserved zinc finger domains when designing peptide antigens

• Absorption techniques:

  • Pre-incubate antibody with recombinant proteins from related family members

  • Use lysates from overexpression systems of potential cross-reactive proteins

• Validation with genetic tools:

  • Compare signals between wild-type and multiple independent At5g50450 mutant lines

  • Include RNAi-silenced lines as additional controls

• Single-cell analysis:

  • Use flow cytometry or single-cell western techniques to validate specificity

  • Compare with fluorescent protein-tagged At5g50450 expression patterns

These strategies address issues similar to those encountered in multimodal single-cell analysis with oligo-conjugated antibodies .

How can At5g50450 antibody be used in conjunction with RNA-seq data to study transcriptional regulation?

For integrative analyses:

• Correlation of protein and transcript levels:

  • Perform RNA-seq on tissues of interest

  • Use At5g50450 antibody for protein quantification in the same tissues

  • Analyze correlation between transcript and protein abundance

  • Identify post-transcriptional regulation events

• ChIP-seq integration:

  • Perform ChIP-seq using At5g50450 antibody

  • Compare binding sites with differentially expressed genes from RNA-seq

  • Identify direct regulatory targets

  • Validate with reporter assays

• Co-expression network analysis:

  • Identify genes with expression patterns similar to At5g50450

  • Use antibody to confirm protein-level correlations

  • Investigate protein-protein interactions within the network

This integrated approach has been successful in identifying functional candidate genes with cis-eQTL in Arabidopsis , and could reveal At5g50450's role in transcriptional networks.

What are common pitfalls when using At5g50450 antibody in various applications?

These troubleshooting approaches address challenges similar to those encountered with other plant antibodies .

How can researchers quantitatively assess At5g50450 protein levels in different tissues or conditions?

For quantitative analysis:

• Fluorescent western blotting:

  • Use fluorescently labeled secondary antibodies

  • Include standard curve with recombinant At5g50450 protein

  • Analyze with imaging systems that provide linear detection range

  • Normalize to multiple housekeeping proteins

• ELISA development:

  • Develop sandwich ELISA using two different At5g50450 antibodies

  • Create standard curve with purified protein

  • Apply to tissue extracts prepared under denaturing conditions

• Quantitative proteomics:

  • Use stable isotope labeling (SILAC or TMT labeling)

  • Immunoprecipitate At5g50450 from different samples

  • Analyze by mass spectrometry for relative quantification

These approaches can be further optimized based on protocols developed for other low-abundance plant transcription factors .

What are the best approaches for generating custom At5g50450 antibodies for specialized research needs?

For custom antibody development:

• Antigen design considerations:

  • Target unique regions outside the conserved MYND zinc finger domain

  • Select sequences with high antigenicity and surface probability

  • Consider both N- and C-terminal regions for epitope selection

  • Evaluate 3D structure predictions to identify exposed regions

• Expression system selection:

  • Bacterial expression: Use E. coli with HIS-tag fusion for peptide antigens (similar to the approach used for AtTIL protein )

  • Eukaryotic expression: Consider insect cells for full-length protein with proper folding

• Validation benchmarks:

  • Test against recombinant protein

  • Compare signals in wild-type vs. multiple At5g50450 mutant lines

  • Evaluate cross-reactivity with other MYND zinc finger proteins

This approach follows successful strategies used for developing antibodies against other plant proteins like AtTIL .

How can At5g50450 antibody be used to investigate protein-protein interactions in zinc finger protein complexes?

For studying protein-protein interactions:

• Proximity labeling techniques:

  • Generate At5g50450-BioID or At5g50450-APEX2 fusion proteins

  • Express in Arabidopsis using appropriate promoters

  • Activate labeling in vivo

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate interactions using At5g50450 antibody

• Pull-down assays:

  • Express GST-tagged At5g50450 in vitro

  • Incubate with plant extracts

  • Identify binding partners by mass spectrometry

  • Confirm interactions using antibodies against specific candidates

These approaches are similar to those used to study ATG6 interactions with NPR1 in Arabidopsis .

What is the relationship between At5g50450 and auxin signaling pathways based on immunoprecipitation studies?

Recent studies suggest potential connections between zinc finger proteins and auxin signaling in Arabidopsis:

• ChIP-seq analysis:

  • Perform ChIP with At5g50450 antibody

  • Identify binding sites in auxin-responsive gene promoters

  • Compare with auxin response element (AuxRE) distribution

  • Validate binding with EMSA using purified protein

• Co-immunoprecipitation with auxin pathway components:

  • Use At5g50450 antibody for Co-IP

  • Probe for interactions with auxin signaling proteins (ARFs, Aux/IAAs)

  • Examine how auxin treatment affects these interactions

  • Validate findings in auxin-signaling mutants

Research into auxin signaling pathways has revealed that NIT2 overexpression increases IAA levels and enhances salt stress tolerance , suggesting zinc finger proteins like At5g50450 might participate in similar regulatory networks.

How can At5g50450 antibody be integrated into multiplexed single-cell protein analysis workflows?

For cutting-edge single-cell protein analysis:

• Oligo-conjugated antibody development:

  • Conjugate At5g50450 antibody with DNA oligonucleotides

  • Optimize conjugation ratio (typically 1:1 to 1:3)

  • Validate specificity after conjugation

  • Determine optimal working concentration (typically 0.1-1 μg/mL)

• Data analysis considerations:

  • Account for background signal in empty droplets

  • Use positive/negative population gating based on At5g50450 mutant controls

  • Normalize UMI counts appropriately

  • Integrate with transcriptomic data when available

These recommendations incorporate lessons from optimizing oligo-conjugated antibodies for multimodal single-cell analysis , adapting them specifically for plant cell systems and At5g50450 detection.

How might AI-based tools improve At5g50450 antibody design and application?

Emerging AI approaches offer new opportunities for At5g50450 antibody research:

• Epitope prediction improvement:

  • Deep learning models can predict optimal epitopes specific to At5g50450

  • Structural prediction tools can identify surface-exposed regions

  • Models can assess cross-reactivity potential with other zinc finger proteins

  • New zero-shot antibody design approaches could generate highly specific binders

• Application optimization:

  • AI can determine optimal antibody concentrations and conditions

  • Image analysis algorithms can enhance immunofluorescence interpretation

  • Machine learning can integrate antibody-based data with other omics datasets

Recent advances in generative AI for de novo antibody design and AI tools for antibody therapies demonstrate the potential for these approaches to revolutionize plant antibody research.

What are best practices for antibody data sharing and reproducibility for At5g50450 research?

To enhance reproducibility in At5g50450 antibody research:

• Comprehensive antibody reporting:

  • Document complete antibody information (source, catalog number, lot)

  • Report all validation experiments performed

  • Include images of full western blots with molecular weight markers

  • Share detailed protocols with all buffer compositions

• Use of antibody repositories:

  • Register antibodies in databases like those listed below :

  Repository Type	Examples	Benefit for At5g50450 Research
  General repositories	Antibody Registry, BenchSci	Standardized antibody identification
  Validation repositories	Antibodypedia	Access to validation data from other researchers
  Application-specific	CiteAb	Find antibodies validated in specific applications

• Protocol standardization:

  • Adopt standardized reporting guidelines

  • Share detailed protocols on platforms like protocols.io

  • Include all negative controls and validation experiments

These practices align with community efforts to improve antibody quality throughout research .

How can researchers integrate multiple antibodies to study At5g50450 in protein complexes regulating stress responses?

For comprehensive analysis of At5g50450 in stress response networks:

• Multiplexed immunoprecipitation:

  • Use At5g50450 antibody in combination with antibodies against known stress-response factors

  • Analyze protein complexes under different stress conditions

  • Compare complex composition between wild-type and mutant plants

  • Correlate with transcriptomic and physiological responses

• Proximity-dependent labeling:

  • Express At5g50450-TurboID fusion in Arabidopsis

  • Apply different stresses (salt, drought, oxidative)

  • Identify stress-specific interaction partners

  • Validate using Co-IP with At5g50450 antibody

• Super-resolution imaging:

  • Use fluorophore-conjugated At5g50450 antibody with antibodies against other factors

  • Apply structured illumination or STORM microscopy

  • Analyze co-localization under different stress conditions

  • Correlate spatial relationships with functional interactions