At1g74820 Antibody

What is AT1G74820 and why is it significant in plant molecular research?

AT1G74820 is a gene in Arabidopsis thaliana that encodes a GLP (Germin-Like Protein) involved in plant stress responses. It has been identified as one of three newly characterized GLP genes in Arabidopsis, alongside AT5G39100 and AT5G61750 . The significance of this gene lies in its potential role in plant defense mechanisms and stress responses. GLPs are known to be involved in basal defense responses conserved among the Gramineae family, with some exhibiting superoxide dismutase (SOD) activity that contributes to hydrogen peroxide (H₂O₂) accumulation during pathogen attack .

The encoded protein participates in stress-responsive pathways, as evidenced by the presence of multiple regulatory elements in GLP gene promoters, including ABA responsive elements (ABRE), anaerobic response elements (ARE), low temperature responsive elements (LTR), and defense-related elements .

How are antibodies against AT1G74820 typically generated for research applications?

Antibodies against AT1G74820 are typically generated using recombinant protein expression systems. Based on common practices observed in the search results, two primary approaches are employed:

• GST-fusion protein immunization: Similar to the approach used for MYC2 antibody generation, researchers produce antibodies by immunizing animals (typically rabbits) with a GST-fusion protein containing specific regions of the target protein . For AT1G74820, this would involve:

  • Cloning the AT1G74820 coding sequence or a specific antigenic region into a GST expression vector

  • Expressing the fusion protein in E. coli

  • Purifying the recombinant protein using glutathione affinity chromatography

  • Immunizing rabbits with the purified protein

  • Collecting and purifying the resulting polyclonal antibodies

• Peptide-based immunization: In cases where full-length protein expression is challenging, synthetic peptides corresponding to unique regions of AT1G74820 can be used as immunogens.

The antibodies are typically purified through affinity purification using the immunogen to ensure specificity .

What are the optimal experimental conditions for Western blot analysis using AT1G74820 antibodies?

When performing Western blot analysis with AT1G74820 antibodies, researchers should consider the following optimized protocol based on similar plant protein antibody applications:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Include reducing agents like DTT (1 mM) to break disulfide bonds

  • For membrane-bound proteins, consider using specialized extraction buffers

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load 20-50 μg of total protein per lane

  • Include positive controls expressing known levels of the target protein

• Antibody dilutions and incubation:

  • Primary antibody (anti-AT1G74820): Use at dilutions between 1:500-1:2,000

  • Secondary antibody: Use anti-rabbit IgG conjugated with HRP at 1:5,000-1:10,000

  • Incubation time: 2 hours at room temperature or overnight at 4°C

• Detection methods:

  • Use enhanced chemiluminescence (ECL) for detection

  • Expected molecular weight may differ from calculated values due to post-translational modifications

• Controls:

  • Include wild-type and knockout/knockdown lines as positive and negative controls

  • Consider boiled versus non-boiled samples to detect potential oligomerization, as observed with other plant proteins

Note that the apparent protein size on Western blots may differ from the calculated molecular weight due to post-translational modifications, similar to what is observed with other plant proteins .

How can researchers validate the specificity of AT1G74820 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For AT1G74820 antibodies, the following validation approaches are recommended:

• Genetic validation:

  • Test antibody reactivity in wild-type plants versus knockout/knockdown mutants of AT1G74820

  • Expect significantly reduced or absent signal in the mutant lines

• Heterologous expression systems:

  • Express recombinant AT1G74820 with an epitope tag (e.g., Flag-tag) in human 293T cells

  • Perform parallel Western blots with both anti-AT1G74820 and anti-tag antibodies

  • The bands should appear at the same molecular weight

• Blocking peptide competition:

  • Pre-incubate the antibody with excess immunizing peptide or protein

  • This should abolish specific signals on Western blots

• Cross-reactivity assessment:

  • Test the antibody against closely related GLP proteins to assess cross-reactivity

  • This is particularly important as AT1G74820 belongs to a family of GLP genes with potential sequence similarity

This multi-faceted validation approach ensures that the observed signals are specific to AT1G74820 and not due to non-specific binding or cross-reactivity with related proteins.

How can AT1G74820 antibodies be utilized in studying plant stress responses?

AT1G74820 antibodies can be instrumental in investigating plant stress responses through several advanced approaches:

• Protein expression profiling under stress conditions:
  Research on related GLP genes shows differential expression under various abiotic stresses. For instance, GLPs from the tandem cluster on chromosome 5 in Arabidopsis showed altered regulation under cold, osmotic, salt, drought, UV-B, and wound stresses . Researchers can use AT1G74820 antibodies to:

  • Track protein accumulation in response to different stresses through Western blotting

  • Compare protein levels with transcript data to identify post-transcriptional regulation

  • Analyze tissue-specific expression patterns under stress conditions

• Subcellular localization changes:

  • Use immunofluorescence microscopy with AT1G74820 antibodies to track changes in protein localization during stress responses

  • Compare normal versus stress conditions to identify potential translocation events

• Protein modification analysis:

  • Use AT1G74820 antibodies to immunoprecipitate the protein under different stress conditions

  • Analyze post-translational modifications (phosphorylation, glycosylation, etc.) that may occur in response to stress

  • Compare modification patterns between stressed and non-stressed plants

Table: Representative GLP Gene Expression Changes Under Abiotic Stresses in Arabidopsis

*Experimental determination of AT1G74820 expression patterns would be valuable for comparison with other GLP family members .

What methodological approaches can be used to study AT1G74820's potential role in disease resistance?

Based on research with related GLP genes, several methodological approaches can be employed to investigate AT1G74820's potential role in disease resistance:

• Pathogen challenge experiments:

  • Challenge Arabidopsis plants with pathogens like Pseudomonas and Phytophthora

  • Use AT1G74820 antibodies to track protein accumulation at different timepoints post-infection

  • Compare wild-type and AT1G74820 knockout/overexpression lines for differences in protein accumulation and disease progression

• H₂O₂ accumulation assays:
  Many GLPs exhibit superoxide dismutase (SOD) activity that contributes to H₂O₂ accumulation during pathogen attack, which is important for defense responses . Researchers can:

  • Measure H₂O₂ accumulation in wild-type versus AT1G74820 mutant plants during pathogen infection

  • Use 3,3'-diaminobenzidine (DAB) staining to visualize H₂O₂ in plant tissues

  • Correlate H₂O₂ levels with AT1G74820 protein levels detected by the antibody

• Elicitor response studies:

  • Treat plants with defense-related elicitors such as Flg22, LPS, HrpZ, or GST-NPP1

  • Use AT1G74820 antibodies to monitor protein accumulation following elicitor treatment

  • Compare with transcriptional data to identify potential post-transcriptional regulation

• Promoter analysis studies:
  Related GLP genes contain multiple defense-related cis-elements in their promoters, including TC-rich repeats responsible for defense and stress (TC-RICH) and wounding and pathogen response elements (W-BOX) . Researchers can:

  • Use reporter assays to correlate promoter activity with protein accumulation

  • Validate protein-level responses using AT1G74820 antibodies

How can researchers combine AT1G74820 antibodies with other molecular tools for functional genomics studies?

Integrating AT1G74820 antibodies with other molecular techniques enables comprehensive functional genomics studies:

• Chromatin Immunoprecipitation (ChIP) studies with transcription factors:
  Several transcription factors, including MYC2, regulate stress-responsive genes in Arabidopsis . Researchers can:

  • Perform ChIP with antibodies against transcription factors like MYC2

  • Analyze binding to the AT1G74820 promoter region

  • Correlate binding events with protein accumulation detected by AT1G74820 antibodies

• Co-immunoprecipitation for protein-protein interaction studies:

  • Use AT1G74820 antibodies to immunoprecipitate the protein and its interacting partners

  • Identify interaction partners through mass spectrometry

  • Validate interactions through reciprocal co-immunoprecipitation

• CRISPR/Cas9-mediated genome editing coupled with antibody detection:

  • Generate precise mutations in AT1G74820 or potential interacting partners

  • Use AT1G74820 antibodies to assess effects on protein stability and accumulation

  • Correlate phenotypic changes with protein levels

• Proximity-dependent labeling approaches:

  • Fuse AT1G74820 to BioID or APEX2

  • Identify proteins in the vicinity of AT1G74820 in living cells

  • Validate proximity using AT1G74820 antibodies in co-localization studies

What are the common challenges when working with AT1G74820 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with AT1G74820 antibodies:

• Cross-reactivity with other GLP family members:

  • Challenge: The GLP gene family contains several members with potential sequence similarity

  • Solution: Perform preliminary tests with recombinant proteins of closely related GLP family members

  • Alternative: Use peptide antibodies raised against unique regions of AT1G74820

• Low expression levels:

  • Challenge: AT1G74820 may be expressed at low levels under basal conditions

  • Solution: Consider enrichment strategies such as immunoprecipitation before Western blotting

  • Alternative: Use stress conditions or elicitors known to induce GLP gene expression

• Post-translational modifications:

  • Challenge: Modifications may affect antibody recognition

  • Solution: Use multiple antibodies targeting different epitopes of AT1G74820

  • Alternative: Consider non-denaturing conditions for detection of native protein

• Oligomerization issues:

  • Challenge: Cupin-family proteins (including GLPs) are known to form homo-multimeric proteins

  • Solution: Compare boiled versus non-boiled samples in Western blots

  • Example: In related proteins, when samples are not boiled prior to SDS-PAGE, oligomeric forms may be observed, whereas boiling results in predominantly monomeric forms

How should researchers design appropriate controls for experiments using AT1G74820 antibodies?

Proper experimental controls are essential for antibody-based research. For AT1G74820 antibodies, consider the following control strategies:

• Genetic controls:

  • Positive control: Wild-type Arabidopsis expressing normal levels of AT1G74820

  • Negative control: AT1G74820 knockout or knockdown lines

  • Overexpression control: Plants overexpressing AT1G74820 to confirm antibody detection limits

• Technical controls for Western blotting:

  • Loading control: Use antibodies against housekeeping proteins like Actin or Tubulin

  • Pre-immune serum control: Use serum collected before immunization to identify non-specific binding

  • Secondary antibody-only control: Omit primary antibody to detect non-specific secondary antibody binding

• Immunolocalization controls:

  • Blocking peptide control: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Fluorophore controls: Include samples with secondary antibody only to detect autofluorescence

  • Subcellular marker co-localization: Use known organelle markers to validate subcellular localization

• Expression system controls:

  • Vector-only control: Express empty vector in heterologous systems (E. coli or human 293T cells)

  • Tagged protein control: Express AT1G74820 with an epitope tag for parallel detection

How can AT1G74820 antibodies contribute to understanding plant-pathogen interactions?

AT1G74820 antibodies can provide valuable insights into plant-pathogen interactions through several innovative approaches:

• Temporal and spatial profiling during infection:

  • Use AT1G74820 antibodies to track protein accumulation at different stages of pathogen infection

  • Perform immunohistochemistry to identify tissue-specific responses

  • Correlate protein localization with sites of pathogen invasion

• Effector-triggered immunity studies:

  • Investigate whether pathogen effectors target AT1G74820 or affect its stability

  • Use co-immunoprecipitation with AT1G74820 antibodies to identify interactions with defense-related proteins

  • Compare protein levels in plants infected with virulent versus avirulent pathogen strains

• Signaling pathway analysis:

  • Use AT1G74820 antibodies to monitor protein accumulation in different defense signaling mutants

  • Investigate how salicylic acid, jasmonic acid, or ethylene signaling affects AT1G74820 protein levels

  • Similar analyses with related GLP genes showed differential regulation under various biotic stresses, including response to elicitors like flagellin fragment 22 (Flg22)

Table: Potential Experimental Design for AT1G74820 Analysis During Pathogen Infection

What are the latest methodological advances in antibody-based plant protein research applicable to AT1G74820 studies?

Recent methodological advances in antibody-based plant protein research can be applied to AT1G74820 studies:

• Single-cell proteomics approaches:

  • Use AT1G74820 antibodies conjugated to heavy metals for mass cytometry

  • Analyze protein abundance at single-cell resolution in plant tissues

  • Identify cell type-specific responses to stress or pathogen infection

• Antibody-based proximity labeling:

  • Conjugate AT1G74820 antibodies to enzymes like APEX2 or TurboID

  • Use in situ to label proteins in proximity to AT1G74820 in intact cells

  • Identify context-specific protein interactions

• Super-resolution microscopy applications:

  • Use fluorescently labeled AT1G74820 antibodies for super-resolution imaging

  • Track protein clustering or membrane association at nanometer resolution

  • Visualize co-localization with defense-related proteins or pathogen structures

• Antibody-based protein stability measurements:

  • Use pulse-chase approaches with AT1G74820 antibodies

  • Determine protein half-life under different stress conditions

  • Identify factors affecting protein stability

Some of these advanced approaches have been employed for studying monoclonal antibodies in other contexts, such as the universal Fabrack-CAR with meditope-enabled monoclonal antibodies (memAbs) described in search result , where sophisticated microscopy techniques tracked interactions over 24-hour periods.