ERD7 Antibody

What is ERD7 and why is it significant for plant stress research?

ERD7 (Early Response to Dehydration 7) is a stress-responsive protein in Arabidopsis thaliana and other plants that accumulates under various abiotic stress conditions including dehydration, cold exposure, salt stress, and ABA treatment. It plays essential roles in both plant stress responses and development. The protein is significant because it modifies membrane lipid composition, which is crucial for cellular adaptation to environmental stresses . Additionally, ERD7 has been identified as a lipid droplet-associated protein in plants undergoing drought stress, suggesting its importance in stress-induced lipid metabolism .

What types of ERD7 antibodies are available for research, and how do they differ?

Currently, polyclonal rabbit antibodies against ERD7 are commercially available. The most characterized anti-ERD7 antibody (AS19 4317) is raised against a KLH-conjugated peptide derived from Arabidopsis thaliana ERD7 (UniProt: O48832, TAIR: AT2G17840) . This antibody has been validated for Western blot applications with a recommended dilution of 1:2000. The antibody specifically recognizes ERD7 protein in Arabidopsis thaliana, appearing as a band of approximately 58 kDa on Western blots (expected molecular weight is 49 kDa) .

What controls should be included when using ERD7 antibody in plant research?

When using ERD7 antibody, researchers should include:

• Negative control: Samples from erd7 knockout/mutant plants should be included to confirm antibody specificity. Studies have used T-DNA insertion mutants (erd7-1) and CRISPR/Cas9-generated mutants (erd7-2) to validate antibody specificity .

• Positive control: Samples from plants exposed to cold stress (4°C for 24h) have been shown to accumulate high levels of ERD7 protein and serve as reliable positive controls .

• Loading control: Standard housekeeping proteins for normalization.

• Cross-reactivity control: Testing potential cross-reactivity with EDN1 and EDN2 (ERD7 homologs) is important, especially when studying ERD7 family functions .

What is the optimal protein extraction protocol for detecting ERD7 in plant tissues?

For optimal detection of ERD7 in Arabidopsis thaliana leaf tissue, the following extraction method has been validated:

• Snap-freeze freshly harvested tissue in liquid nitrogen and grind into a fine powder.

• Extract total protein with an extraction buffer containing:

  • 50 mM Tris-HCl pH 8.0

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.1% SDS

  • 0.5% Na-Deoxycholate

  • 2 mM PMSF

  • 2 mM DTT

Alternative extraction buffer composition for immunodetection of tagged ERD7:

• 100 mM Tris-HCl pH 7.5

• 150 mM NaCl

• 10% (v/v) glycerol

• 0.1% (v/v) Tween-20

• 1 mM PMSF

• Protease Inhibitor Cocktail

• Denature samples with Laemmli buffer containing 2% β-mercaptoethanol at 70°C for 10 minutes.

• Separate proteins on 10% SDS-PAGE for optimal resolution of the 58 kDa ERD7 band .

What are the recommended Western blot conditions for ERD7 detection?

For optimal Western blot detection of ERD7:

• Transfer conditions: Semi-dry transfer to PVDF membrane for 1 hour has shown good results .

• Blocking: Use 5% non-fat milk in TBS-T for 1 hour at room temperature with agitation.

• Primary antibody incubation: Dilute anti-ERD7 antibody 1:2000 in TBS-T with 1% milk and incubate overnight at 4°C with agitation.

• Washing: Rinse briefly with TBS-T, then wash three times for 5-10 minutes each in TBS-T at room temperature with agitation.

• Secondary antibody: Use anti-rabbit IgG-HRP at 1:25,000 dilution in TBS-T with 1% milk for 1 hour at room temperature.

• Detection: Develop with chemiluminescent detection reagent for 5 minutes .

How can researchers overcome common technical challenges when detecting ERD7 protein?

Common challenges and solutions include:

• Background signal: A band of lower molecular weight may appear in the soluble fraction of both wild-type and erd7 mutant plants. This is a background signal and should not be confused with ERD7 . To differentiate, always include proper controls and focus on the microsomal fraction where authentic ERD7 is predominantly found.

• Low signal intensity: ERD7 protein levels vary significantly with stress conditions. For stronger signals, expose plants to cold (4°C for 24h), which induces higher accumulation than salt or ABA treatments .

• Non-specific binding: Increasing blocking time or concentration (up to 5% milk) and adding more washing steps can help reduce non-specific binding.

• Degradation: ERD7 may be susceptible to degradation. Always prepare fresh samples, keep them cold, and add sufficient protease inhibitors to all buffers.

How does ERD7 protein accumulation differ under various stress conditions?

Research has demonstrated differential accumulation of ERD7 protein under various stress conditions:

• Cold stress: ERD7 shows strongest accumulation after exposure to 4°C for 24 hours, with a clear 58 kDa band detected by Western blotting .

• Salt stress: NaCl treatment induces ERD7 protein accumulation but to a lesser extent than cold treatment .

• ABA treatment: Application of abscisic acid also induces ERD7 protein, but the response is not as robust as with cold stress .

• Drought stress: ERD7 has been identified in lipid droplets isolated from drought-stressed Arabidopsis leaves, indicating its role in drought response mechanisms .

This differential accumulation suggests distinct roles for ERD7 in various stress response pathways, with potentially more significant functions in cold stress adaptation.

What insights has ERD7 antibody provided into subcellular localization during stress responses?

ERD7 antibody has revealed important insights into the protein's subcellular distribution:

• Membrane association: Subcellular fractionation followed by Western blotting with anti-ERD7 antibody showed that native ERD7 predominantly localizes to the microsomal fraction in cold-stressed plants .

• Lipid droplet association: Proteomics analysis of lipid droplets isolated from drought-stressed Arabidopsis leaves identified ERD7 as a lipid droplet-associated protein, which was later confirmed using fluorescent protein-tagged ERD7 and imaging techniques .

• Dual localization pattern: ERD7 appears to have a dual localization pattern, with distribution between lipid droplets and the cytosol, but not uniform distribution to all membrane compartments .

• ER association: While not uniformly distributed throughout the ER membrane, ERD7 family proteins show some colocalization with ER markers, suggesting a partial association with ER membranes .

These findings suggest that ERD7 may shuttle between different cellular compartments during stress responses, potentially playing roles in membrane remodeling and lipid metabolism.

How can researchers study the relationship between ERD7 and membrane lipid composition?

Advanced approaches to study ERD7's role in membrane lipid composition include:

• Lipid binding assays: Recombinant MBP-ERD7 fusion proteins can be used in protein-lipid overlay assays with pre-spotted phospholipids on nitrocellulose membranes. This approach has revealed that ERD7 interacts with negatively charged phospholipids including cardiolipin, phosphatidic acid, and phosphoinositides .

• Membrane fluidity analysis: Comparing membrane fluidity between wild-type and erd7 mutant plants using fluorescence anisotropy techniques can reveal ERD7's role in maintaining membrane properties during stress. Previous research has shown reduced membrane fluidity in erd7 mutant lines .

• Lipidomics approaches: Quantitative lipidomics comparing lipid profiles of wild-type and mutant plants under control and stress conditions can identify specific lipid changes dependent on ERD7 function.

• Cold-induced phospholipid changes: Analysis of cold-induced changes in phosphoinositides, particularly PIP2, in wild-type versus erd7 mutant plants can provide insights into ERD7's role in stress-induced membrane remodeling .

What experimental approaches can be used to investigate ERD7's role in lipid droplet dynamics?

Sophisticated experimental approaches to study ERD7's role in lipid droplet biology include:

• Domain mapping: Creating deletion constructs of ERD7 fused to fluorescent tags (like mCherry) can identify domains required for lipid droplet targeting. Research has shown that the C-terminal senescence domain (amino acids 258-426) is both necessary and sufficient for lipid droplet targeting .

• Live-cell imaging: Time-lapse fluorescence microscopy of plants expressing fluorescently-tagged ERD7 during stress induction can reveal dynamic changes in ERD7 localization and lipid droplet formation.

• Lipid droplet isolation: Biochemical isolation of lipid droplets from stress-treated plants followed by Western blotting can quantify ERD7 recruitment to this compartment under different conditions.

• Stress-induced lipid droplet changes: Comparing lipid droplet numbers, size, and distribution in wild-type versus erd7 mutant plants under stress conditions using microscopy and lipid staining (e.g., BODIPY or Monodansylpentane) .

What approaches can be used to study ERD7 protein interactions during stress responses?

Advanced methods to investigate ERD7 protein interactions include:

• Yeast two-hybrid screening: This approach has identified numerous potential ERD7-interacting proteins involved in stress responses, including some that have been identified in lipid droplet proteomes .

• Co-immunoprecipitation: Using anti-ERD7 antibody to pull down protein complexes from stressed plant tissues, followed by mass spectrometry, can identify native interaction partners.

• Bimolecular fluorescence complementation (BiFC): This technique can visualize and confirm protein-protein interactions in planta and determine in which subcellular compartment these interactions occur.

• Proximity labeling: BioID or APEX2 fused to ERD7 can identify proximal proteins in living cells through biotinylation, providing spatial information about the ERD7 interactome.

• Comparative interactomics: Comparing ERD7 interactomes under different stress conditions can reveal stress-specific interaction networks.

How can ERD7 antibody be used to analyze mutant and transgenic lines?

ERD7 antibody provides several important applications for genetic analysis:

• Verification of knockout lines: Anti-ERD7 can confirm the absence of ERD7 protein in T-DNA insertion mutants (erd7-1) or CRISPR/Cas9-generated mutants (erd7-2) .

• Analysis of genetic redundancy: Due to functional redundancy with homologs (EDN1 and EDN2), antibody-based detection of ERD7 is crucial for understanding compensation mechanisms in single versus higher-order mutants .

• Evaluation of stress-responsive expression: Comparative analysis of ERD7 protein levels in different genetic backgrounds under stress can reveal regulatory factors controlling ERD7 expression.

• Confirmation of transgene expression: For complementation studies or overexpression lines, anti-ERD7 can verify protein expression levels.

What insights can be gained by comparing ERD7 and its homologs using antibody-based approaches?

Comparative analysis of ERD7 family proteins can provide valuable insights:

• Expression pattern differences: Using specific antibodies against ERD7, EDN1, and EDN2 allows comparison of their accumulation patterns under various stress conditions to identify specialized functions.

• Subcellular localization differences: Despite high sequence similarity (>62% identity), ERD7 family members may have distinct subcellular localization patterns that can be detected using immunolocalization with specific antibodies .

• Functional redundancy analysis: In knockout mutant backgrounds, antibody detection can reveal compensatory upregulation of remaining family members.

• Domain function analysis: The senescence domains of different family members have been shown to confer targeting to either lipid droplets or mitochondria, which can be analyzed using domain-specific antibodies .

How can researchers design experiments to resolve the apparent contradiction between membrane and lipid droplet localization of ERD7?

To address the dual localization pattern of ERD7, researchers can:

• Stress-dependent localization analysis: Systematically analyze ERD7 localization under different stresses and timepoints using subcellular fractionation followed by Western blotting with anti-ERD7 antibody.

• Co-localization studies: Perform detailed co-localization analysis with markers for different membrane compartments (ER, plasma membrane) and lipid droplets under controlled conditions.

• Live-cell imaging: Use fluorescently-tagged ERD7 combined with time-lapse imaging to track potential translocation events during stress induction.

• Mutational analysis: Create point mutations in the senescence domain that specifically disrupt either membrane or lipid droplet binding without affecting the other, and analyze using anti-ERD7 for native protein or anti-tag antibodies for mutated versions.

• Lipid environment manipulation: Alter cellular lipid composition pharmacologically or genetically and assess impacts on ERD7 localization using antibody-based detection methods.

Table 1. Experimental Conditions for Optimal ERD7 Detection by Western Blot

Table 2. Comparative Analysis of ERD7 Accumulation Under Different Stress Conditions