EMB1187 Antibody

What is the biological function of the EMB1187 protein targeted by this antibody?

EMB1187 (At2g26830) functions as a probable ethanolamine kinase (EC 2.7.1.82) involved in phospholipid biosynthesis, specifically catalyzing the initial step in phosphatidylethanolamine biosynthesis. This enzyme belongs to the choline/ethanolamine kinase family and plays a crucial role in membrane biogenesis in Arabidopsis thaliana. Research indicates that related family members such as CEK4, a plasma membrane-localized isoform, catalyze the rate-limiting step in phosphatidylcholine (PtdCho) biosynthesis and are essential for proper embryo development. When designing experiments targeting EMB1187, researchers should consider its subcellular localization and enzymatic function in phospholipid metabolism pathways.

How can researchers validate the specificity of EMB1187 antibody for experimental applications?

Validating antibody specificity is critical, especially considering that up to one-third of antibodies exhibit nonspecific binding to unintended targets . For EMB1187 antibody, researchers should implement multiple validation approaches:

• Western blot analysis comparing wild-type and EMB1187 knockout/knockdown samples

• Immunoprecipitation followed by mass spectrometry

• Immunofluorescence with appropriate negative controls

• Pre-absorption tests with recombinant EMB1187 protein

• Cross-reactivity assessment with related choline/ethanolamine kinase family members

This multi-method validation approach helps ensure experimental results accurately reflect EMB1187 biology rather than artifacts from nonspecific binding. Documentation of validation procedures should be maintained and included in research publications to support reproducibility.

What are the optimal storage conditions for maintaining EMB1187 antibody activity?

The EMB1187 antibody should be stored according to manufacturer specifications to maintain its activity and specificity. Based on similar research-grade antibodies, the recommended storage conditions include:

• Short-term storage (1 month): 2-8°C under sterile conditions after reconstitution

• Long-term storage (6-12 months): -20 to -70°C under sterile conditions after reconstitution

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw

The buffer composition (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% ProClin 300 as preservative) helps maintain antibody stability. Researchers should monitor antibody performance over time, as even properly stored antibodies may gradually lose activity. When designing long-term studies, consider creating a validation schedule to ensure consistent antibody performance throughout the research timeline.

What controls should be included when using EMB1187 antibody in complex plant tissue samples?

When studying EMB1187 in plant tissues, particularly during embryo development, comprehensive controls are essential for result interpretation:

Additionally, researchers should consider tissue-specific and developmental timing factors in experimental design. EMB1187's role in embryo development suggests potential expression variations across different plant tissues and developmental stages. Parallel experiments with antibodies against other phospholipid biosynthesis enzymes can provide contextual understanding of pathway regulation.

How can researchers optimize immunodetection of EMB1187 in subcellular fractionation studies?

Detecting EMB1187 in subcellular fractions presents technical challenges due to its association with membrane components and potential low abundance. Optimization strategies include:

• Membrane protein extraction optimization:

  • Test multiple detergent combinations (e.g., CHAPS, NP-40, Triton X-100)

  • Evaluate detergent concentration effects on antigen epitope preservation

  • Consider crosslinking approaches for preserving protein-protein interactions

• Signal enhancement techniques:

  • Implement tyramide signal amplification for immunofluorescence

  • Optimize blocking conditions to reduce background (test BSA vs. milk proteins)

  • Evaluate primary antibody concentration ranges (0.5-5 μg/mL) and incubation times

• Subcellular fraction validation:

  • Confirm fraction purity using established organelle markers

  • Verify membrane integrity during fractionation procedures

  • Assess phospholipid profiles of fractions to correlate with EMB1187 localization

Researchers should systematically document optimization parameters to establish reproducible protocols. Creating standardized procedures helps distinguish genuine biological variations from technical artifacts when comparing EMB1187 localization across different experimental conditions.

How should researchers interpret discrepancies between EMB1187 protein levels and corresponding gene expression data?

Discrepancies between protein and mRNA levels are common in biological systems and require careful interpretation. For EMB1187, consider these analytical approaches:

• Temporal relationship analysis:

  • Track both mRNA and protein levels across a detailed time course

  • Consider time lags between transcription and protein accumulation

  • Document half-life differences between mRNA and protein

• Post-transcriptional regulation assessment:

  • Investigate potential microRNA regulation of EMB1187 mRNA

  • Analyze RNA-binding protein interactions with EMB1187 transcripts

  • Examine alternative splicing patterns that might affect antibody epitope recognition

• Post-translational modification evaluation:

  • Assess phosphorylation states that might affect antibody binding

  • Investigate potential proteolytic processing of EMB1187

  • Consider protein complex formation that might mask antibody epitopes

When publishing results showing such discrepancies, clearly present both datasets with appropriate statistical analyses and discuss potential biological explanations rather than dismissing either dataset as erroneous. This approach promotes a more complete understanding of EMB1187 regulation in plant phospholipid biosynthesis.

To what extent can EMB1187 antibody be used for cross-species studies of ethanolamine kinases?

When considering cross-species applications of the EMB1187 antibody, researchers should evaluate epitope conservation:

• Sequence homology analysis:

  • Perform multiple sequence alignments of ethanolamine kinases across species

  • Identify conserved regions that might contain the antibody epitope

  • Calculate percent identity in potential epitope regions

• Empirical cross-reactivity testing:

  • Conduct Western blot analysis with protein extracts from multiple plant species

  • Include positive controls (Arabidopsis) alongside test species

  • Document band patterns and molecular weights across species

• Epitope mapping considerations:

  • If known, compare the antibody epitope sequence across species

  • Consider synthesizing peptides representing homologous regions for competition assays

  • Evaluate structural conservation using homology modeling

How can EMB1187 antibody contribute to functional analysis of phospholipid biosynthesis pathway mutants?

The EMB1187 antibody serves as a valuable tool for investigating mutants in phospholipid biosynthesis:

• Pathway component relationships:

  • Compare EMB1187 protein levels across mutants of related pathway enzymes

  • Assess compensatory expression changes in response to pathway disruptions

  • Correlate EMB1187 localization with membrane composition alterations

• Structure-function analyses:

  • Use the antibody to detect truncated or modified forms of EMB1187 in point mutants

  • Evaluate how mutations affect protein stability and subcellular localization

  • Correlate antibody epitope accessibility with predicted structural changes

• Developmental phenotype correlations:

  • Track EMB1187 expression in embryo-defective mutants at different developmental stages

  • Correlate protein levels with phospholipid profile changes and developmental abnormalities

  • Document tissue-specific expression patterns in relation to phenotypic manifestations

This application is particularly relevant given that EMB1187 (and related family member CEK4) has been implicated as essential for Arabidopsis embryo development, making the antibody a crucial tool for understanding phospholipid metabolism in plant development.

What specialized protocols enable successful EMB1187 detection in seeds and developing embryos?

Detecting EMB1187 in seeds and embryonic tissues presents unique challenges due to high lipid content, protective structures, and potential low abundance. Specialized approaches include:

• Sample preparation optimization:

  • Test modified extraction buffers containing lipase inhibitors

  • Evaluate cryosectioning vs. paraffin embedding for tissue preservation

  • Consider laser capture microdissection for stage-specific embryo isolation

• Signal-to-noise enhancement:

  • Implement antigen retrieval methods optimized for lipid-rich tissues

  • Test extended blocking procedures with specialized blocking agents

  • Consider using amplification systems like biotin-streptavidin for immunohistochemistry

• Multi-modal validation:

  • Correlate antibody signals with in situ hybridization patterns

  • Compare results with fluorescently-tagged EMB1187 in transgenic lines

  • Validate findings using mass spectrometry-based proteomics

These methodological adaptations can help overcome the inherent challenges of detecting enzymes involved in phospholipid metabolism in complex and lipid-rich plant reproductive tissues.

What strategies can resolve weak or inconsistent EMB1187 detection signals?

When encountering weak or variable EMB1187 detection, consider this systematic troubleshooting approach:

• Antibody validation:

  • Verify antibody activity using dot blot or ELISA with recombinant protein

  • Consider obtaining a new antibody lot if degradation is suspected

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

• Sample preparation refinement:

  • Evaluate different extraction methods to improve protein solubilization

  • Test protease inhibitor cocktail formulations to prevent degradation

  • Consider native vs. denaturing conditions based on epitope accessibility

• Detection system optimization:

  • Compare different secondary antibodies or detection reagents

  • Evaluate signal amplification methods appropriate for the application

  • Adjust exposure times and image acquisition parameters

• Protocol standardization:

  • Implement strict temperature control during incubation steps

  • Standardize washing procedures to reduce background variability

  • Document all procedural details to identify sources of variation

Creating a detailed troubleshooting decision tree specific to your experimental system can help systematically resolve detection issues while maintaining scientific rigor .

How should researchers interpret and address potential cross-reactivity with other ethanolamine/choline kinase family members?

Given that EMB1187 belongs to the choline/ethanolamine kinase family, cross-reactivity is a legitimate concern:

• Computational prediction:

  • Conduct in silico epitope analysis against all family members

  • Identify unique and shared sequence regions across the kinase family

  • Create a cross-reactivity risk assessment based on sequence homology

• Experimental verification:

  • Test antibody against recombinant proteins of related family members

  • Perform immunoprecipitation followed by mass spectrometry to identify all captured proteins

  • Use genetic knockout lines of family members to assess signal specificity

• Complementary approaches:

  • Implement siRNA/CRISPR knockdown of EMB1187 to confirm signal reduction

  • Correlate antibody signals with orthogonal detection methods

  • Consider developing epitope-tagged versions for highly specific detection

How can EMB1187 antibody studies integrate with emerging phospholipidomics approaches?

The integration of EMB1187 antibody-based studies with phospholipidomics creates powerful research opportunities:

• Correlative multimodal analysis:

  • Combine immunolocalization data with lipid mass spectrometry imaging

  • Correlate EMB1187 levels with quantitative changes in phosphatidylethanolamine species

  • Integrate protein-lipid interaction studies with enzyme localization patterns

• Systems biology integration:

  • Incorporate EMB1187 protein levels into metabolic flux models of phospholipid biosynthesis

  • Correlate enzyme distribution with computational predictions of membrane composition

  • Develop multi-scale models connecting molecular interactions to cellular phenotypes

• Technological synergies:

  • Implement proximity labeling techniques using EMB1187 antibodies to identify interacting partners

  • Combine super-resolution microscopy with specific antibody detection for nanoscale localization

  • Develop biosensors incorporating EMB1187-binding fragments for live monitoring of enzyme dynamics

This integrated approach provides a more comprehensive understanding of phospholipid metabolism regulation in plant development and stress responses, extending beyond what can be achieved through antibody detection alone.

What remaining questions about EMB1187 function could be addressed through improved antibody-based approaches?

Despite current knowledge, several fundamental questions about EMB1187 remain addressable through enhanced antibody applications:

• Regulatory mechanisms:

  • How do post-translational modifications regulate EMB1187 activity and localization?

  • What protein complexes incorporate EMB1187 under different developmental conditions?

  • How do membrane microenvironments influence EMB1187 enzymatic activity?

• Developmental dynamics:

  • How does EMB1187 distribution change during critical developmental transitions?

  • What signals trigger EMB1187 relocalization during cellular responses?

  • How does EMB1187 abundance correlate with specific embryo developmental defects?

• Stress response roles:

  • How does EMB1187 respond to environmental stresses affecting membrane integrity?

  • What role does EMB1187 play in phospholipid remodeling during temperature stress?

  • How do plant pathogens affect EMB1187 localization and activity?

Addressing these questions requires developing more sophisticated antibody-based approaches, potentially including phosphorylation-specific antibodies, conformation-sensitive antibodies, and multiplexed detection systems for simultaneous tracking of multiple pathway components.