At5g65370 Antibody

What is the At5g65370 gene and its encoded protein in Arabidopsis thaliana?

At5g65370 is a gene locus in Arabidopsis thaliana that encodes a protein containing the Epsin N-terminal homology (ENTH) domain . This protein is part of the cellular machinery involved in vesicular trafficking and membrane dynamics. The ENTH domain is a conserved structural motif that functions in clathrin-mediated endocytosis and other membrane-related transport processes. In Arabidopsis, this protein plays roles in intracellular trafficking pathways that are important for plant development and stress responses .

What is the relationship between At5g65370 and membrane trafficking?

The At5g65370 gene product is related to vesicular trafficking processes in Arabidopsis cells. According to gene expression analyses, it functions alongside other trafficking-related proteins such as dynamin-like proteins and clathrin-binding proteins . Research indicates that these proteins are part of a coordinated system for endosomal protein trafficking, potentially affecting protein transport between cellular compartments. The expression of At5g65370 appears to be regulated in response to certain cellular conditions, suggesting its involvement in adaptive trafficking responses .

How can I verify the specificity of the At5g65370 antibody for my experiments?

To verify antibody specificity:

• Perform Western blot analysis using both wild-type and At5g65370 knockout or knockdown plant tissues

• Include appropriate positive and negative controls in immunolocalization experiments

• Conduct pre-adsorption tests with the purified antigen

• Compare staining patterns with previously published localization data

• Use multiple antibodies targeting different epitopes of the same protein when possible

The antibody available from commercial sources (e.g., CSB-PA863790XA01DOA) should be validated using these approaches before proceeding with detailed experimental applications .

What are the recommended protocols for using At5g65370 antibody in immunolocalization studies?

For optimal immunolocalization results with At5g65370 antibody:

• Tissue fixation: Use 4% paraformaldehyde in PBS for 2-4 hours at room temperature

• Sectioning: Prepare 5-10 μm sections of paraffin-embedded or cryo-preserved tissue

• Antigen retrieval: Perform citrate buffer (pH 6.0) treatment if necessary

• Blocking: Incubate sections with 3-5% BSA or normal serum for 1 hour

• Primary antibody: Dilute At5g65370 antibody (CSB-PA863790XA01DOA) to 1:100-1:500 and incubate overnight at 4°C

• Secondary antibody: Use fluorophore-conjugated or HRP-conjugated antibodies at manufacturer-recommended dilutions

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

• Controls: Always run parallel experiments with pre-immune serum or secondary antibody only

These protocols should be optimized based on your specific tissue type and experimental conditions.

How can At5g65370 antibody be used to study vesicular trafficking under stress conditions?

The At5g65370 protein has been implicated in cellular trafficking processes that may be altered during stress responses. To study these dynamics:

• Subject plant samples to relevant stress conditions (salt, drought, temperature)

• Harvest tissues at different time points after stress application

• Perform immunoprecipitation with At5g65370 antibody to isolate protein complexes

• Analyze co-precipitated proteins by mass spectrometry to identify stress-specific interaction partners

• Conduct immunofluorescence microscopy to track changes in At5g65370 localization during stress

• Compare trafficking patterns between wild-type and mutant plants under stress conditions

Research has shown that vesicular trafficking components, including ENTH domain-containing proteins, can show altered expression or localization patterns during stress responses, particularly in salt stress where Na+/H+ exchangers like AtNHX1 are involved .

What methodologies can be employed to study the interaction between At5g65370 and other vesicular trafficking components?

To investigate protein-protein interactions involving At5g65370:

• Co-immunoprecipitation: Use At5g65370 antibody to pull down protein complexes from plant extracts, followed by Western blotting or mass spectrometry

• Proximity ligation assay (PLA): Detect in situ interactions between At5g65370 and suspected partner proteins

• Bimolecular Fluorescence Complementation (BiFC): Express At5g65370 and potential interactors as fusion proteins with split fluorescent protein fragments

• Yeast two-hybrid screening: Identify novel interaction partners from Arabidopsis cDNA libraries

• FRET/FLIM analysis: Measure energy transfer between fluorescently tagged proteins to confirm direct interactions

These approaches can reveal functional relationships between At5g65370 and other trafficking components such as clathrin, dynamin-like proteins, and small GTPases that have been implicated in related pathways .

How does the expression and localization of At5g65370 change in response to altered cellular ion homeostasis?

Studies of ion transport mutants, particularly those affecting Na+/H+ exchangers like AtNHX1, provide insights into At5g65370's potential roles in cellular responses to ionic stress:

• In AtNHX1 knockout lines, genes encoding vesicular trafficking proteins show altered expression patterns, suggesting coordinated regulation

• To study At5g65370 responses to ion homeostasis disruption:

  • Compare At5g65370 localization between wild-type and ion transport mutants using immunofluorescence

  • Monitor At5g65370 expression levels in response to various ion stresses using quantitative immunoblotting

  • Assess the impact of At5g65370 mutation on cellular ion content using inductively coupled plasma mass spectrometry (ICP-MS)

  • Investigate colocalization with ion transporters during stress using dual-labeling immunofluorescence

The relationship between vesicular trafficking components and ion transporters appears physiologically significant, as demonstrated by altered expression of trafficking-related genes in ion transport mutants .

What autophagy-related processes might involve At5g65370, and how can they be investigated?

While direct evidence linking At5g65370 to autophagy is not explicitly stated in the search results, vesicular trafficking proteins often intersect with autophagy pathways. To investigate potential connections:

• Compare At5g65370 localization with autophagosome markers during nutrient starvation or stress

• Assess At5g65370 expression levels in autophagy-deficient mutants (e.g., atg5, atg7)

• Determine whether At5g65370 knockout affects autophagy flux using standard autophagy monitoring techniques

• Investigate potential interaction between At5g65370 and ATG proteins using co-immunoprecipitation

• Analyze the impact of autophagy inducers and inhibitors on At5g65370 distribution and function

The autophagy machinery has been shown to be crucial for antigen presentation and immune responses in dendritic cells , suggesting that membrane trafficking components like At5g65370 might similarly participate in autophagy-related vesicular processes in plants.

What are the common technical challenges when working with At5g65370 antibody, and how can they be addressed?

Researchers may encounter several challenges when using At5g65370 antibody:

• Background signal: Optimize blocking conditions (5% BSA or milk protein) and increase washing duration/frequency

• Low signal strength: Try antigen retrieval methods, increase antibody concentration, or extend incubation time

• Non-specific binding: Pre-absorb antibody with total protein extract from knockout mutants

• Inconsistent results: Standardize sample preparation protocols and antibody handling procedures

• Epitope masking: Test multiple fixation protocols that preserve epitope accessibility

• Antibody degradation: Aliquot stock solutions and avoid repeated freeze-thaw cycles

For optimal storage and handling, follow manufacturer recommendations for the specific antibody preparation (CSB-PA863790XA01DOA) .

How can At5g65370 antibody be validated in knockout or knockdown lines?

Thorough validation of antibody specificity using genetic resources is crucial:

• Obtain confirmed knockout or knockdown lines for At5g65370 from repositories such as ABRC or NASC

• Perform RT-PCR and qRT-PCR to confirm reduced transcript levels in these lines

• Run Western blots comparing wild-type and mutant samples, looking for absence or reduction of the target band

• Conduct immunolocalization in parallel on wild-type and mutant tissues using identical protocols

• Consider complementation experiments where the gene is reintroduced to confirm restoration of antibody signal

• Document all validation steps meticulously for publication and reproducibility

This validation process ensures that any signal detected with the antibody represents the true At5g65370 protein rather than cross-reactivity with other proteins.

How should researchers design experiments to study At5g65370's role in response to environmental stresses?

For robust experimental designs investigating At5g65370 function during stress:

• Include multiple time points (early, intermediate, late responses) after stress application

• Compare multiple stress types (ionic, osmotic, oxidative, temperature) to identify specific vs. general responses

• Use both physiological and molecular readouts to correlate At5g65370 dynamics with plant responses

• Incorporate genetic approaches (knockout, overexpression, point mutations) to establish causality

• Design rescue experiments with wild-type or modified versions of At5g65370

• Include relevant controls for each stress treatment

• Analyze multiple tissues and developmental stages

Gene expression studies have shown that vesicular trafficking components, including potential At5g65370-related pathways, are differentially regulated during salt stress responses , suggesting experimental approaches should consider ionic stress responses.

What bioinformatic approaches can complement At5g65370 antibody-based studies?

Integrative bioinformatic analyses can enhance antibody-based research on At5g65370:

• Protein domain analysis to predict functional motifs and potential interaction surfaces

• Co-expression network analysis using publicly available transcriptomic datasets

• Phylogenetic comparisons with ENTH domain proteins across plant species

• Protein structural modeling to predict epitope accessibility and protein function

• Analysis of promoter elements to understand transcriptional regulation

• Mining of phosphoproteomic data to identify potential regulatory phosphorylation sites

• Prediction of subcellular localization and trafficking signals

Integrating these computational approaches with experimental data from antibody-based studies provides a more comprehensive understanding of At5g65370 function and regulation within cellular networks.

What are the emerging research directions for At5g65370 antibody applications in plant biology?

Future research utilizing At5g65370 antibody may focus on:

• High-resolution imaging techniques (super-resolution microscopy, electron microscopy) to precisely localize At5g65370 within membrane microdomains

• Proteomics approaches to identify dynamic interactomes under different conditions

• Cell-specific and tissue-specific expression analysis using immunohistochemistry

• Comparative studies across ecotypes and related species to understand evolutionary conservation

• Integration with other trafficking components to build comprehensive models of plant vesicular transport

• Potential biotechnological applications in improving plant stress tolerance

• Investigation of pharmaceutical compounds targeting vesicular trafficking to modulate plant stress responses