ERD14 Antibody

What is ERD14 and why is it important in research?

ERD14 (Early Response to Dehydration 14) is a plant dehydrin protein highly expressed in Arabidopsis thaliana cells during heat and dehydration stress. It functions as an intrinsically disordered stress protein (IDSP) with chaperone-like properties, capable of protecting other proteins during stress conditions. ERD14 is characterized by conserved sequence motifs, including K-segments, S-segments, and H-segments, which play differential roles in its protective function .

ERD14 is particularly valuable as a model protein for studying:

• Molecular mechanisms of plant stress response

• Protein chaperone activity of intrinsically disordered proteins

• Structure-function relationships in conditionally disordered proteins

• Cellular protection mechanisms against abiotic stresses

What are the best methods for ERD14 antibody production?

Production of high-quality ERD14 antibodies requires careful consideration of several factors:

• Protein Purification: ERD14 can be purified using a heat treatment method that exploits its intrinsic disorder and heat stability:

  • Express ERD14 in BL21(DE3) cells with 0.7 mM IPTG induction

  • Lyse cells in buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, protease inhibitors

  • Boil the lysate for 20 minutes to remove globular proteins (ERD14 remains soluble)

  • Perform ion-exchange chromatography using DEAE Sepharose followed by MonoQ columns

• Antibody Production and Purification:

  • Immunize mice with purified recombinant ERD14

  • Isolate polyclonal IgG using protein G column chromatography

  • Purify ERD14-specific antibodies using CNBr-activated Sepharose 4B resin with immobilized ERD14

• Validation: Confirm antibody specificity through Western blot analysis using both recombinant protein and plant tissue extracts

What are the optimal conditions for using ERD14 antibody in immunological techniques?

When working with ERD14 antibody, consider these methodological recommendations:

Western Blot:

• Sample preparation: Note that ERD14 is enriched in disorder-promoting amino acids (Gly, Ser, Lys, Glu, Pro) and depleted in order-promoting amino acids (Trp, Cys)

• This compositional bias leads to weak binding to Coomassie blue dye and low molar extinction coefficient at 280 nm

• Use appropriate antibody dilutions (typically 1:1000 to 1:5000) in PBS-T buffer

• Longer exposure times may be necessary due to the protein's unusual amino acid composition

Storage and Handling:

• Store antibody in lyophilized form when possible

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Upon receipt, store immediately at recommended temperature (typically -20°C)

How can ERD14 antibody be used to study protein-protein interactions?

ERD14 antibodies can be employed in multiple techniques to investigate protein-protein interactions:

Cross-linking Coupled with Immunoprecipitation:

• Cross-link proteins using glutaraldehyde (25% v/v) with 5N HCl

• Incubate samples at 25°C for 15 minutes

• Analyze via SDS-PAGE followed by western blotting with anti-ERD14 antibodies

• This method has successfully shown ERD14 interaction with GSTF9

Pull-down Assays with Biotinylated ERD14:

• Express biotinylated ERD14 in bacterial systems (e.g., AVB101 strain)

• Cross-link with dithiobis(succinimidyl propionate) (DSP) at 2 mM at 30°C for 7 minutes

• Pull down using Streptavidin-MAG-Sepharose

• Analyze bound proteins by SDS-PAGE and mass spectrometry (LC-MS/MS)

Microscale Thermophoresis (MST):

• Label ERD14 with fluorescent dye (e.g., Atto 647 NHS ester)

• Titrate with increasing concentrations of potential binding partners

• Perform MST with 20% LED power and 40% MST power

• This method has been used to characterize ERD14 interactions with GSTF9 and catalase

What are the challenges in detecting ERD14 using antibodies given its intrinsically disordered nature?

Several methodological challenges arise when working with ERD14 antibodies due to the protein's disordered structure:

Research has shown that ERD14 tends to oligomerize at higher concentrations (75 μM and above), forming complexes of approximately 50 kDa, 70 kDa, and 200 kDa. This concentration-dependent behavior should be considered when interpreting antibody-based detection results .

How can ERD14 antibody be used to investigate subcellular localization during stress responses?

ERD14 antibodies are valuable tools for studying the protein's dynamic localization during stress conditions:

Immunofluorescence Microscopy Protocol:

• Fix plant tissues with 4% paraformaldehyde

• Perform antigen retrieval if necessary

• Block with appropriate serum (typically 5% BSA or normal goat serum)

• Incubate with anti-ERD14 primary antibody

• Apply fluorescently-labeled secondary antibody

• Counterstain with DAPI for nuclear visualization

Research Findings on Localization:

• Studies have shown that ERD14 is primarily localized in the cytoplasm during heat stress

• This cytoplasmic localization argues against membrane protection as its primary function

• Instead, evidence suggests ERD14 functions through promiscuous protein binding and protection

For studying ERD14's role in brassinosteroid signaling, researchers have generated ERD14-GFP fusion constructs under both native (ERD14pro:ERD14-GFP) and constitutive (35Spro:ERD14-GFP) promoters to monitor localization dynamics in relation to BRL3 receptor trafficking .

What techniques can be used to study the structural dynamics of ERD14 in vivo?

In-cell NMR Spectroscopy:

• Transform BL21 Star (DE3) pLysS cells with constructs expressing wild-type or modified ERD14

• Grow cells in LB medium, then transfer to M9 minimal medium with 15N-labeled ammonium chloride

• For assignment, use both 15N-labeled ammonium chloride and 13C-labeled glucose

• Induce expression with 1.6 mM IPTG for 3 hours at 37°C

• Pellet cells and resuspend in 15N-free M9 minimal medium with 10% D2O

• Record 15N-HSQC spectra on a Bruker DRX 500 MHz spectrometer at 277K

This technique has revealed that while ERD14 is largely disordered in cells, its K-segments transiently sample helical conformations and engage in partner binding. Other regions (S-segment and linking regions) remain disordered and unbound even during functional interactions .

How can ERD14 antibody be used to study stress protection mechanisms?

ERD14 antibodies can be employed to investigate cellular protection mechanisms through these methodological approaches:

Cell Viability Assays:

• Express ERD14 (wild-type or variants) in E. coli cells

• Subject cells to heat stress (e.g., 50°C for 15 minutes)

• Measure survival rates compared to control cells

• Use antibodies to confirm and quantify ERD14 expression levels

• Normalize protection effects to expression levels

Findings from Such Studies:

• ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress

• The protection is mainly achieved by protecting the cellular proteome

• Functional studies with deletion mutants revealed differential contributions of ERD14's conserved segments to its protective function:

  • Deletion of the C-terminal K-segment (ΔKc) reduced protection significantly (DSR: 19.6%)

  • Deletion of the S-segment had the most pronounced effect (DSR: 23.4%)

What is known about ERD14's interaction with BRL3 and how can antibodies help study this relationship?

Recent research has revealed ERD14's role in chaperoning the brassinosteroid receptor BRL3:

Key Experimental Approaches:

• Co-immunoprecipitation with anti-ERD14 antibodies to pull down BRL3-ERD14 complexes

• Subcellular localization studies using ERD14-GFP and BRL3-GFP constructs

• Phenotypic analysis of erd14 mutants and their effects on BRL3 signaling

Research Findings:

• ERD14 regulates BRL3 subcellular localization and activity

• In erd14 mutants, BRL3-GFP shows aberrant internalization and reduced abundance

• The chaperoning function of ERD14 appears similar to other receptor-like kinase chaperones such as LORELEI-LIKE GLYCOSYL PHOSPHATIDYL INOSITOL-ANCHORED PROTEIN 1 (LLG1) and DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY)

• ERD14 may facilitate the simultaneous association of BRL3 with multiple signaling partners during abiotic stress

How can ERD14 antibody be used in comparative studies across different plant species?

When studying ERD14 across different plant species, consider these methodological approaches:

Cross-species Reactivity Assessment:

• Perform sequence alignment of ERD14 homologs across species of interest

• Identify conserved epitopes that may be recognized by the antibody

• Test antibody reactivity on protein extracts from different species via Western blot

• Validate specificity using knockout/knockdown lines when available

Immunohistochemistry Protocol for Cross-species Studies:

• Use standardized tissue preparation methods across species

• Include appropriate negative controls (pre-immune serum, secondary antibody only)

• Include peptide competition assays to confirm specificity

• When possible, complement with mRNA expression data (qPCR or RNA-seq)

What are common pitfalls when working with ERD14 antibody and how can they be addressed?

How can the sensitivity and specificity of ERD14 antibody detection be optimized?

Sensitivity Enhancement Strategies:

• Use signal amplification systems (e.g., tyramide signal amplification)

• Employ more sensitive detection substrates for Western blots

• Optimize antibody concentration through titration experiments

• Use recombinant protein standards to establish detection limits

Specificity Optimization Approaches:

• Pre-absorb antibody with related dehydrin proteins

• Use peptide competition assays to confirm signal specificity

• Include appropriate negative controls (samples from knockout lines)

• Consider developing antibodies against unique regions rather than conserved motifs

Validation Through Multiple Methods:

• Compare results from multiple detection techniques (Western blot, immunohistochemistry, ELISA)

• Correlate protein detection with mRNA expression data

• Confirm functional relationships through genetic approaches (mutant analysis)

What considerations are important when designing experiments to study ERD14 post-translational modifications?

ERD14, like other dehydrins, may undergo various post-translational modifications that affect its function and interactions:

Methodological Approaches:

• Phosphorylation Analysis:

  • Use phospho-specific antibodies if available

  • Perform phosphatase treatments to confirm phosphorylation

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

  • Validate with mass spectrometry

• Other Modifications:

  • Consider potential methylation, acetylation, or ubiquitination

  • Use modification-specific enrichment techniques before antibody detection

  • Employ mass spectrometry for comprehensive modification mapping

Experimental Design Considerations:

• Include appropriate controls for modification state (e.g., phosphatase-treated samples)

• Consider the impact of stress conditions on modification status

• Account for potential epitope masking by modifications

• When possible, compare wild-type ERD14 with mutants lacking modification sites

How might ERD14 antibodies contribute to understanding plant adaptation to climate change?

ERD14 antibodies can play crucial roles in climate change adaptation research:

• Comparative Studies Across Ecotypes:

  • Analyze ERD14 expression and modification patterns in plants from diverse climatic regions

  • Correlate protein abundance with stress tolerance phenotypes

  • Investigate natural variation in ERD14 structure and function

• Stress Response Time-course Studies:

  • Monitor ERD14 expression, localization, and interaction dynamics during progressive stress exposure

  • Identify critical thresholds for protective responses

  • Develop predictive models for plant stress tolerance

• Crop Improvement Applications:

  • Screen germplasm collections for optimal ERD14 expression patterns

  • Validate stress protection mechanisms in major crop species

  • Support breeding programs with molecular markers for enhanced stress tolerance

What novel methodologies might enhance ERD14 antibody-based research?

Emerging technologies that could advance ERD14 antibody research include:

• Super-resolution Microscopy:

  • Apply techniques like STORM or PALM for nanoscale localization of ERD14

  • Investigate co-localization with interaction partners at subcellular resolution

  • Examine dynamic changes in protein distribution during stress response

• Single-cell Proteomics:

  • Detect cell-to-cell variation in ERD14 expression and localization

  • Correlate with single-cell transcriptomics data

  • Identify specialized cell types critical for stress protection

• Protein Engineering Approaches:

  • Develop conformation-specific antibodies to capture different structural states

  • Create bifunctional antibody constructs for proximity labeling

  • Engineer nanobodies for in vivo imaging applications