ERF011 Antibody

What is ERF011 and what is its functional role in plants?

ERF011 (also known as DEAR1, ATERF#011, or CEJ1) is a member of the DREB subfamily A-5 of ERF/AP2 transcription factor family in Arabidopsis thaliana. It functions as a transcriptional repressor involved in defense and freezing stress responses. There are 16 members in this subfamily including RAP2.1, RAP2.9, and RAP2.10 . The protein contains an EAR (ERF Amphiphilic Repression) motif which enables its transcriptional repression function .

Which applications are ERF011 antibodies typically validated for?

According to product specifications, commercially available ERF011 antibodies are generally validated for Western Blot (WB) applications with typical recommended dilutions of 1:1000-1:2000 . Some antibodies may be suitable for additional applications such as ELISA, but this varies by manufacturer and specific antibody clone.

What is the expected molecular weight of ERF011 protein?

The expected molecular weight of DEAR1/ERF011 is approximately 17 kDa as indicated in product specifications . When performing Western blot analysis, this is the primary band size researchers should expect to observe when using a specific ERF011 antibody.

What are the recommended storage conditions for ERF011 antibodies?

For optimal maintenance of antibody activity, the storage recommendations are:

• Lyophilized form: Store at -20°C to -70°C

• After reconstitution: Store at 4°C for up to 1 month

• For long-term storage after reconstitution: Aliquot and store at -20°C

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

How should I reconstitute lyophilized ERF011 antibody?

Reconstitution should be performed as follows:

• Briefly centrifuge the vial before opening to collect all material at the bottom

• Add 150 μL of sterile water to the lyophilized antibody

• Gently mix to ensure complete dissolution

• Once reconstituted, the antibody can be aliquoted to avoid repeated freeze-thaw cycles

What are the proper positive controls for validating ERF011 antibody specificity?

For proper validation, use recombinant protein containing the peptide used for immunization. For example, in antibody validation studies:

• Use 2.5 ng, 10 ng and 25 ng of recombinant protein containing the immunization peptide

• Run on 12% SDS-PAGE

• Transfer to nitrocellulose membrane for 1 hour

• Block with 5% skim milk at RT or 4°C for 1 hour

• Incubate with primary antibody at 1:1000 dilution overnight at 4°C

• Use appropriate HRP-conjugated secondary antibody at 1:10000 dilution

How can I minimize cross-reactivity when using ERF011 antibody?

Given that the ERF family contains multiple similar members, cross-reactivity is a significant concern. Take these steps to minimize it:

• Use antibodies generated against unique epitopes (not conserved domains)

• Perform peptide competition assays to confirm specificity

• Include appropriate negative controls (tissues not expressing ERF011)

• Consider using ERF011 knockout/mutant lines as negative controls

• Verify results using multiple techniques (e.g., WB combined with qRT-PCR)

How can I use ERF011 antibody to study its interaction with other transcription factors?

ERF011/ERF11 has been shown to interact with other transcription factors and proteins. To study these interactions:

• Co-immunoprecipitation (Co-IP) approach:

  • Prepare plant tissue lysates under non-denaturing conditions

  • Use ERF011 antibody immobilized on protein A/G beads

  • Elute bound complexes and analyze by Western blot with antibodies against suspected interaction partners

  • Verify interactions using reciprocal Co-IP

• For ERF-DELLA protein interactions specifically:

  • Use yeast two-hybrid assays complemented with Co-IP

  • Perform transient expression assays in plant systems

  • These methods have successfully demonstrated interactions between ERF11 and DELLA proteins, showing how ERF11 enhances GA signaling by antagonizing DELLA proteins

What strategies can I employ to study ERF011's role in transcriptional regulation during stress responses?

To investigate ERF011's function in transcriptional regulation during stress:

• ChIP (Chromatin Immunoprecipitation) approach:

  • Cross-link proteins to DNA in plant tissues exposed to stress conditions

  • Immunoprecipitate chromatin using ERF011 antibody

  • Analyze bound DNA regions by qPCR or sequencing

  • Focus on promoters of stress-responsive genes

• Time-course analysis:

  • Expose plants to specific stresses (e.g., mannitol-induced stress)

  • Collect samples at multiple timepoints (5min to 3h)

  • Use ERF011 antibody to track protein levels and post-translational modifications

  • Correlate with gene expression changes of target genes

How can I distinguish between ERF011 and other closely related ERF family members in my experiments?

This is a critical challenge due to the high sequence homology within the ERF family. Advanced approaches include:

• Using a proximity ligation assay (PLA) combined with specific antibodies:

  • Similar to the approach used for ERCC1 isoforms, combine an ERF011-specific antibody with antibodies that recognize other domains

  • This allows detection of the complete protein rather than just epitopes

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of ERF011

  • Compare binding patterns across different experimental conditions

  • Differences in reactivity patterns can help distinguish between family members

What might cause false positives when using ERF011 antibody, and how can I address them?

False positives are a significant concern with antibodies against transcription factor families. The causes and solutions include:

• Cross-reactivity with related ERF family members:

  • Verify antibody specificity using knockout mutants

  • Perform peptide competition assays

  • Compare results with mRNA expression data

• Non-specific binding:

  • Increase blocking time and concentration

  • Optimize antibody dilution (test range between 1:500-1:2000)

  • Include detergents in wash buffers

  • Use more stringent washing conditions

Why might I observe different results with ERF011 antibody under different stress conditions?

ERF011/ERF11 expression and activity are highly regulated by environmental conditions. Variation in results may be due to:

• Diurnal expression patterns:

  • ERF011 transcript levels follow a diurnal pattern, being low in the morning, accumulating until evening, and decreasing again in early morning

  • Time tissue collection consistently based on this pattern

• Stress-specific regulation:

  • ERF011 is differentially regulated by various stresses

  • Hormone treatments (especially ethylene and jasmonate) affect ERF011 levels

  • Control for these variables in experimental design

How can I validate that my observed band truly represents ERF011 and not a cross-reactive protein?

Rigorous validation is essential due to documented cases of antibody cross-reactivity. For conclusive validation:

• Perform peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots with and without peptide competition

  • Specific bands should disappear in the peptide-blocked sample

• Use genetic controls:

  • Compare wild-type samples with ERF011 knockout/knockdown lines

  • Specific bands should be absent or reduced in knockout/knockdown samples

• Correlation with mRNA expression:

  • Compare protein detection with mRNA levels across conditions

  • Discrepancies may indicate cross-reactivity or post-transcriptional regulation

How does ERF011/ERF11 function in the balance between plant growth and stress response?

ERF011/ERF11 plays a complex role in growth-stress balance:

• Antagonistic relationship with ERF6:

  • ERF6 acts as a transcriptional activator inducing stress response genes

  • ERF11 molecularly counteracts ERF6 by competing for the same target promoters

  • This creates a negative feedback loop that fine-tunes stress responses

  • The ERF6-ERF11 antagonism helps maintain balance between growth and defense

• Dual role in growth regulation:

  • ERF11 plays a dual role in promoting internode elongation by:
    a) Inhibiting ethylene biosynthesis (repressing ACS gene expression)
    b) Activating gibberellin biosynthesis and signaling pathways

  • This facilitates appropriate growth responses during stress recovery

What technical advances might improve ERF011 antibody specificity and research applications?

Based on advancements in antibody technology, several approaches could enhance ERF011 research:

• Development of heterodimer-specific antibodies:

  • Similar to approaches used for ERCC1/XPF, antibodies could be developed that specifically recognize ERF011 in complex with its interacting partners

  • This would allow more specific detection of functional protein complexes

• Proximity ligation assay (PLA) for specific detection:

  • Combining antibodies that recognize different epitopes in a PLA format

  • This approach has been successful for distinguishing between functionally different isoforms of other proteins

  • Could be adapted to distinguish ERF011 from closely related family members

• Advanced epitope selection:

  • Targeting truly unique regions of ERF011 to minimize cross-reactivity

  • Employing computational approaches to identify epitopes with minimal homology to other ERF family members