At5g60610 Antibody

What is At5g60610 and why is it significant in plant research?

At5g60610 is a gene locus in Arabidopsis thaliana that appears in research contexts involving plant-nematode interactions. Based on current literature, it may be involved in ubiquitination pathways, similar to other Arabidopsis proteins that interact with plant pathogens. Particularly, it may function in a manner similar to UPL3 (Homology to E6-AP C-Terminus-type ubiquitin E3 ligase), which plays a role in the Arabidopsis response to nematode infections like Globodera pallida .

The protein encoded by At5g60610 is likely significant in plant defense mechanisms and may contribute to understanding plant-nematode interactions that are critical for agricultural research and crop protection strategies.

How are antibodies against Arabidopsis proteins like At5g60610 typically generated?

Antibodies against Arabidopsis proteins are typically generated through the following methodological approach:

• Protein Expression: The target protein (or fragment) is expressed and purified

• Immunization: BALB/c mice are commonly immunized with the purified protein or protein complex

• Hybridoma Generation: Spleen cells are fused with myeloma cells to create hybridomas

• Selection: Positive clones are selected through ELISA screening

• Clone Development: Monoclonal hybridoma cell lines are developed through limiting dilution

• Antibody Purification: Typically using Protein G affinity chromatography

For example, antibodies against plant proteins like Actin-7 are generated by immunizing BALB/c mice with Arabidopsis thaliana Actin-7, followed by hybridoma selection and antibody purification .

What experimental validation is necessary for confirming At5g60610 antibody specificity?

Thorough validation of antibody specificity for At5g60610 should include:

• Western blot analysis showing single band of expected size in wild-type plants

• Absence or reduced signal in knockout/knockdown lines (e.g., T-DNA insertion lines)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-reactivity testing against closely related proteins

• Pre-absorption controls with recombinant antigen

Multiple validation approaches are critical since plant proteins often belong to gene families with high sequence similarity. For example, anti-Actin-7 antibodies were validated using multiple applications (WB, ELISA, IF) and it is recommended to use all three monoclonal antibodies in first-time, qualitative experimental setups to determine which is most suitable for specific experiments .

What are the recommended storage and handling conditions for plant antibodies like At5g60610?

Proper storage and handling are crucial for maintaining antibody activity:

For antibodies provided as cell culture supernatant, storage at -80°C is recommended for periods longer than one month, similar to the anti-Rhamnogalacturonan I antibody .

How should I optimize co-immunoprecipitation (co-IP) experiments with At5g60610 antibodies?

For successful co-IP experiments with plant proteins like At5g60610:

• Expression System Selection:

  • Use Nicotiana benthamiana leaves for transient expression of tagged proteins

  • Consider using tagged versions (HA-tag, Myc-GFP-tag) of the target proteins

• Protocol Optimization:

  • Use appropriate buffer conditions (typically containing 150-300mM NaCl, 1% NP-40, protease inhibitors)

  • Include proper negative controls (empty vectors, unrelated proteins)

  • Confirm expression levels before immunoprecipitation

• Detection Strategy:

  • Use sensitive detection methods for western blot analysis

  • Consider sequential or simultaneous probing with different antibodies

This approach has been successful in demonstrating interactions between plant proteins and pathogen effectors, as shown in studies of StUPL3 interaction with GpRbp-1 in plant cells .

What methods are effective for studying At5g60610 localization in plant cells?

To effectively determine subcellular localization:

• Immunofluorescence Microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Optimize permeabilization conditions for plant cell walls and membranes

  • Use appropriate blocking solution (3-5% BSA or normal serum)

  • Incubate with primary antibody at optimized dilution

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain with organelle markers (e.g., DAPI for nucleus)

• Bimolecular Fluorescence Complementation (BiFC):

  • Clone At5g60610 into a BiFC vector containing half of a fluorescent protein

  • Clone potential interacting partners into complementary vectors

  • Co-express in plant cells (N. benthamiana leaves or protoplasts)

  • Analyze fluorescence restoration using confocal microscopy

This approach was successful in studies showing nuclear localization of protein interactions, as demonstrated for StUPL3 in plant cells .

How can I address non-specific binding when using antibodies in plant tissues?

Non-specific binding is a common challenge with plant tissues. Methodological approaches to minimize this include:

• Blocking Optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time (from 1h to overnight)

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce hydrophobic interactions

• Antibody Dilution:

  • Titrate antibody concentrations (typically starting at 1:500-1:2000)

  • Pre-absorb antibody with plant extract from knockout/mutant lines

• Washing Conditions:

  • Increase number of washes (5-6 washes of 10 minutes each)

  • Add higher salt concentration (up to 500mM NaCl) to reduce ionic interactions

  • Consider adding 0.05% SDS to washing buffer for western blots

• Secondary Antibody Selection:

  • Use highly cross-adsorbed secondary antibodies

  • Consider using secondary antibodies specifically tested for plant applications

These approaches help minimize background while maintaining specific signal detection.

How should I interpret changes in At5g60610 protein levels during experimental treatments?

For accurate interpretation of protein level changes:

• Quantification Methods:

  • Use densitometry software (ImageJ/FIJI) for western blot quantification

  • Normalize to appropriate loading controls (total protein or housekeeping proteins)

  • Apply statistical analysis across biological replicates (minimum n=3)

• Biological Relevance Assessment:

  • Compare changes with transcript levels (qRT-PCR)

  • Correlate with phenotypic changes in wild-type vs. mutant plants

  • Confirm with orthogonal methods (e.g., mass spectrometry-based quantification)

• Common Pitfalls to Avoid:

  • Overexposure of western blot leading to signal saturation

  • Using inappropriate loading controls affected by treatment

  • Failure to account for protein degradation during sample preparation

With proper controls and quantification, antibody-based protein level measurements can provide valuable insights into At5g60610 regulation during stress responses or developmental processes.

How can I use At5g60610 antibodies to study plant-pathogen interactions?

For studying plant-pathogen interactions:

• Infection Time Course Analysis:

  • Monitor At5g60610 protein levels during infection progression

  • Compare susceptible vs. resistant plant varieties

  • Correlate with pathogen colonization/reproduction rates

• Co-localization Studies:

  • Perform dual immunolabeling with pathogen effector proteins

  • Analyze potential relocalization of At5g60610 during infection

  • Use confocal microscopy for spatial resolution of interactions

• Protein-Protein Interaction Analysis:

  • Use co-immunoprecipitation to identify pathogen targets

  • Perform proximity labeling experiments (BioID, APEX)

  • Validate interactions with in vitro binding assays

These approaches can reveal whether At5g60610 is manipulated by pathogen effectors similar to other plant defense components. For example, the effector GpRbp-1 from Globodera pallida has been shown to interact with the potato UPL3 homolog, suggesting a potential virulence mechanism targeting ubiquitination pathways .

What approaches can be used to study post-translational modifications of At5g60610?

To investigate post-translational modifications:

• Ubiquitination Analysis:

  • Co-express HA-tagged ubiquitin with At5g60610 in plant cells

  • Immunoprecipitate At5g60610 and probe for ubiquitin modification

  • Use deubiquitinating enzyme inhibitors during extraction

  • Analyze polyubiquitination patterns via western blotting

• Phosphorylation Studies:

  • Use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Perform immunoprecipitation followed by phospho-specific antibody detection

  • Consider phosphatase treatments as negative controls

  • Use mass spectrometry to identify phosphorylation sites

• Glycosylation Analysis:

  • Treat samples with PNGase F to remove N-linked glycans

  • Compare mobility shifts before and after treatment

  • Use lectin blotting to confirm glycosylation status

Similar approaches have been used to demonstrate E3 ubiquitin ligase activity of StUPL3 in planta, where exogenous HA-Ub was used for polyubiquitination detection .

How can I develop functional assays to study At5g60610 activity in relation to plant immunity?

To develop functional assays:

• Gene Silencing/Knockout Approaches:

  • Generate CRISPR/Cas9 knockout lines

  • Use RNAi or artificial microRNA for knockdown

  • Analyze phenotypic consequences during pathogen infection

  • Compare with wild-type responses using quantitative metrics

• Complementation Studies:

  • Transform knockout lines with wild-type or mutated At5g60610

  • Assess restoration of function using infection assays

  • Analyze domain-specific contributions to protein function

• Activity Assays:

  • If At5g60610 has enzymatic activity, develop in vitro assays

  • For E3 ligases, measure ubiquitination of putative substrates

  • Monitor subcellular changes in response to infection

For example, Arabidopsis mutants with reduced expression of UPL3 (upl3-5) showed subtle changes in susceptibility to cyst nematodes, demonstrating how genetic approaches complement antibody-based studies in understanding protein function in immunity .

How can I correlate antibody-based findings with transcriptomic data for At5g60610?

For effective data integration:

• Expression Pattern Analysis:

  • Compare protein levels (western blot) with transcript abundance (RNA-seq/qPCR)

  • Analyze tissue-specific or condition-specific differences

  • Identify potential post-transcriptional regulation mechanisms

• Co-expression Network Construction:

  • Identify genes with similar expression patterns to At5g60610

  • Correlate with protein interaction data from immunoprecipitation studies

  • Build functional networks incorporating both datasets

• Promoter Analysis:

  • Use ChIP-seq data to identify transcription factors regulating At5g60610

  • Correlate with protein-level changes during stress responses

This integrative approach provides a comprehensive understanding of At5g60610 regulation and function in plant biology, similar to transcriptomic studies of Arabidopsis responses to reduced UPL3 expression .

What bioinformatic resources are available for predicting epitopes and antibody specificity for At5g60610?

Key bioinformatic tools and approaches include:

When developing new antibodies against At5g60610, these resources help design immunogens targeting unique, accessible epitopes, minimizing cross-reactivity with related proteins.