At5g66270 Antibody

What is At5g66270 and why is it significant in plant molecular biology?

At5g66270 encodes a Zinc finger CCCH domain-containing protein 68 in Arabidopsis thaliana. It belongs to the CCCH zinc finger family, which includes 68 members in Arabidopsis . This protein contains specific zinc finger motifs characterized by the C-X8-C-X5-C-X3-H pattern and is involved in nucleic acid binding . Its significance stems from its anther-specific expression pattern (stages 8-12), suggesting a role in reproductive development . The protein's precise function remains under investigation, but CCCH zinc finger proteins are generally involved in RNA processing, metabolism, and stress responses in plants.

How are At5g66270 antibodies developed and validated?

At5g66270 antibodies are typically developed using synthetic peptides corresponding to specific epitopes of the target protein. Similar to other plant antibodies, this process involves:

• Peptide design and synthesis for immunization

• Immunization of host animals (commonly rabbits)

• Collection of antisera and purification of antibodies

• Validation through multiple techniques:

  • Western blotting against plant extracts

  • Immunohistochemistry on plant tissues

  • Testing for cross-reactivity with related proteins

  • Verification in knockout mutants (e.g., T-DNA insertion lines)

Rigorous validation is critical as demonstrated by research on other plant antibodies that showed specific bands in wild-type Arabidopsis but absence in corresponding knockout mutants .

What are the recommended protocols for using At5g66270 antibody in Western blotting?

For optimal Western blot results with At5g66270 antibody:

• Sample preparation:

  • Homogenize Arabidopsis tissues in ice-cold extraction buffer (0.1 M TES, pH 7.8, 0.2 M NaCl, 1 mM EDTA, 2% β-mercaptoethanol, 1 mM PMSF)

  • Separate proteins on SDS-PAGE and transfer to membrane (Hybond-C recommended)

• Immunoblotting protocol:

  • Block membrane in TTBS (TBS plus 0.05% Tween 20) with 5% nonfat milk for 2 hours

  • Incubate with primary At5g66270 antibody (typically 1:1000 dilution) for 2 hours

  • Wash three times with TTBS

  • Incubate with secondary antibody for 1 hour

  • Detect using either Amplified Alkaline Phosphatase assay or ECL detection system

• Controls:

  • Include wild-type and At5g66270 knockout/mutant samples

  • Consider using tissues with known expression patterns (anther tissue is recommended)

How can At5g66270 antibody be used for localization studies in plant tissues?

For subcellular localization studies using At5g66270 antibody:

• Immunohistochemistry (IHC):

  • Fix tissue samples in 4% paraformaldehyde

  • Embed in paraffin or prepare cryosections

  • Perform antigen retrieval if necessary

  • Block with serum and incubate with At5g66270 antibody

  • Detect using fluorescence-conjugated secondary antibodies

• Immunoelectron microscopy:

  • Fix tissue samples in glutaraldehyde and embed in resin

  • Prepare ultrathin sections and mount on grids

  • Incubate with At5g66270 antibody followed by gold-conjugated secondary antibody

  • Examine using transmission electron microscopy to determine precise subcellular localization

• Confocal microscopy with fluorescent protein fusions:

  • Create GFP or RFP fusions with At5g66270

  • Transform Arabidopsis plants or transiently express in plant cells

  • Use antibody in co-localization studies to confirm native protein distribution

How can researchers verify the specificity of commercial At5g66270 antibodies?

To verify antibody specificity:

• Genetic controls:

  • Compare immunoblot signals between wild-type and At5g66270 knockout/mutant plants

  • Use CRISPR/Cas9-generated knockout lines as negative controls

  • Complement mutant lines to restore antibody reactivity

• Biochemical validation:

  • Perform peptide competition assays by pre-incubating antibody with the immunizing peptide

  • Conduct immunoprecipitation followed by mass spectrometry to confirm target identity

  • Compare reactivity patterns across different tissues with known expression patterns

• Cross-reactivity assessment:

  • Test against recombinant At5g66270 protein

  • Examine reactivity with closely related CCCH zinc finger proteins

  • Document any non-specific bands and their molecular weights

This approach is essential as demonstrated by cases like anti-glucocorticoid receptor antibody clone 5E4, which showed significant cross-reactivity with unintended proteins .

What are common issues encountered with At5g66270 antibody and how can they be resolved?

Common issues and their solutions include:

How can At5g66270 antibody be employed in chromatin immunoprecipitation (ChIP) studies?

For ChIP applications with At5g66270 antibody:

• Protocol optimization:

  • Cross-link plant tissues with 1% formaldehyde

  • Extract and sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with At5g66270 antibody (typically 5-10 μg per reaction)

  • Include appropriate controls (IgG, input DNA)

  • Reverse cross-links and purify DNA

  • Analyze by qPCR or sequencing

• Critical considerations:

  • Verify antibody specificity for ChIP by testing in pilot experiments

  • Optimize chromatin fragmentation for CCCH zinc finger proteins

  • Include known binding sites as positive controls if available

  • Consider cell-type specific ChIP if At5g66270 has tissue-specific expression

• Data analysis approach:

  • Compare enrichment over IgG controls and input

  • Use peak calling software appropriate for plant ChIP-seq data

  • Identify DNA binding motifs using tools like MEME suite

What approaches can be used to study protein-protein interactions involving At5g66270?

To investigate protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Extract proteins from appropriate tissues (anthers recommended)

  • Immunoprecipitate using At5g66270 antibody

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm interactions by reverse Co-IP or bimolecular fluorescence complementation

• Proximity labeling approaches:

  • Create fusion proteins with BioID or TurboID

  • Express in Arabidopsis under native or tissue-specific promoters

  • Use At5g66270 antibody to confirm expression and localization

  • Purify biotinylated proteins and identify by mass spectrometry

• Yeast two-hybrid screening:

  • Use At5g66270 as bait to screen Arabidopsis cDNA libraries

  • Confirm interactions in planta using Co-IP with At5g66270 antibody

  • Consider domain-specific interactions given the CCCH zinc finger structure

How does At5g66270 expression pattern correlate with its proposed biological function?

The At5g66270 gene shows anther-specific expression during stages 8-12 of flower development , suggesting a specialized role in male reproductive development. Consider these aspects when interpreting antibody data:

• Developmental context:

  • Stage 8: Microsporogenesis begins, locules become visible

  • Stages 9-10: Meiosis occurs, tapetum development is critical

  • Stages 11-12: Microspore maturation and pollen development

• Cell-type specificity:

  • Determine if expression is in tapetum, microsporocytes, or other anther tissues

  • Correlate expression with specific developmental events in anthers

  • Compare with other anther-specific CCCH proteins for functional redundancy

• Functional implications:

  • CCCH proteins often regulate RNA metabolism

  • Expression pattern suggests possible roles in:

    • Meiotic progression in microsporocytes

    • Tapetum development or programmed cell death

    • RNA processing during pollen development

How can researchers integrate At5g66270 antibody data with transcriptomic and proteomic analyses?

For comprehensive multi-omics integration:

• Data correlation approaches:

  • Compare protein levels (antibody detection) with transcript levels (RNA-seq)

  • Look for post-transcriptional regulation if discrepancies exist

  • Correlate with developmental or stress-responsive transcriptome datasets

• Network analysis:

  • Place At5g66270 in protein-protein interaction networks

  • Identify co-expressed genes and protein partners

  • Use antibody-based co-IP data to validate predicted interactions

• Functional genomics integration:

  • Correlate antibody-detected protein levels with phenotypes in mutants

  • Perform ChIP-seq to identify direct targets if At5g66270 binds DNA/RNA

  • Use tissue-specific proteomics to complement antibody localization data

What controls are essential when using At5g66270 antibody for immunoprecipitation?

Essential controls for immunoprecipitation include:

• Genetic controls:

  • Wild-type vs. At5g66270 knockout mutant tissues

  • Complemented lines expressing tagged At5g66270 for validation

• Technical controls:

  • Input sample (pre-IP) to assess starting material

  • IgG control from same species as primary antibody

  • Beads-only control to identify non-specific binding

  • Pre-immune serum control if available

• Validation approaches:

  • Western blot of IP sample with the same or different At5g66270 antibody

  • Mass spectrometry confirmation of immunoprecipitated protein

  • Peptide competition assay to demonstrate specificity

How should researchers adapt protocols when working with At5g66270 antibody in different plant species or tissues?

When adapting protocols across species or tissues:

• Cross-species applications:

  • Verify epitope conservation through sequence alignment

  • Perform initial validation in the new species

  • Adjust extraction buffers based on tissue composition

  • Consider using higher antibody concentrations initially

• Tissue-specific adaptations:

  • For reproductive tissues: Use stage-specific collections

  • For vegetative tissues: Check expression databases first

  • For recalcitrant tissues: Modify extraction buffers to account for secondary metabolites

• Protocol optimization:

  • Adjust fixation times for immunohistochemistry based on tissue density

  • Modify extraction buffers for different subcellular compartments

  • Consider tissue-specific blocking agents to reduce background

  • Optimize antibody concentrations for each new tissue/species context