ERF095 Antibody

What is ERF095 and what role does it play in plant biology?

ESE1 (also known as ERF095 or ETHYLENE AND SALT INDUCIBLE 1) is a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family. This subfamily includes 18 members including ATERF-1, ATERF-2, and ATERF-5 . Like other members of the ETS family of transcription factors, ERFs regulate cell proliferation and differentiation through a highly conserved DNA-binding domain that recognizes the sequence GGAA/T . ERF095 specifically plays crucial roles in plant stress responses, particularly to ethylene and salt stress, as indicated by its full name.

What types of ERF095 antibodies are available for research applications?

Current research primarily utilizes rabbit polyclonal antibodies targeting the C-terminal region of ERF095. For example, commercial antibodies like PHY7856S are developed using KLH-conjugated synthetic peptides (17 amino acids from the C-terminal section) derived from Arabidopsis thaliana ESE1 (AT3G23220) . These polyclonal antibodies offer advantages in detecting native proteins across multiple applications, particularly Western blot analysis where the expected molecular weight is approximately 16 kDa .

What are the most rigorous methods for validating ERF095 antibody specificity?

The gold standard for antibody validation employs genetic approaches using knockout (KO) cell lines rather than orthogonal approaches. Research shows that 89% of antibodies validated using genetic strategies demonstrate genuine specificity, compared to only 80% of those validated through orthogonal methods . For ERF095 antibody validation, researchers should:

• Generate ERF095 knockout lines using CRISPR-Cas9 or other gene editing techniques

• Perform side-by-side Western blot comparison between wild-type and knockout samples

• Test antibody reactivity against recombinant ERF095 protein at multiple concentrations (e.g., 2.5 ng, 10 ng, 25 ng)

• Verify specificity across intended applications (Western blot, immunofluorescence, etc.)

How does the choice of immunogen affect ERF095 antibody performance?

The immunogen selection critically impacts antibody performance. For ERF095, antibodies generated against C-terminal epitopes may perform differently than those targeting the DNA-binding domain or other regions. When evaluating ERF095 antibodies, researchers should:

• Assess whether the immunogen represents a unique or conserved region within the ERF family

• Consider potential post-translational modifications that might affect epitope accessibility

• Determine if the epitope is accessible in native versus denatured protein conformations

• Evaluate potential cross-reactivity with other ERF family members through sequence alignment analysis

What controls are essential when using ERF095 antibody in immunoblotting experiments?

Proper controls are critical for valid immunoblotting results with ERF095 antibodies. As emphasized in multiple studies, rigor and reproducibility depend heavily on experimental design—including the use of rigorous controls . Essential controls include:

• Negative antibody controls:

  • Pre-immune serum from the rabbit used to generate the antibody

  • Commercial rabbit serum not exposed to the immunogen

  • Isotype-matched control antibodies

• Positive controls:

  • Independent antibody raised against different ERF095 epitopes

  • Recombinant ERF095 protein at known concentrations

  • Samples with verified ERF095 expression

• Tissue controls:

  • Samples from tissues known to express ERF095

  • Samples from tissues or cells with ERF095 knocked out or knocked down

How should single-stain controls be incorporated when using ERF095 antibody in fluorescence-based applications?

For fluorescence applications using ERF095 antibodies, single-stain controls are absolutely essential. According to flow cytometry best practices, single-stain controls must be run every time an experiment is performed . Researchers should:

• Include single-stain controls for ERF095 antibody and all other fluorophores in multi-parameter experiments

• Run controls on the same day as experimental samples, as "variations in antibody staining, fluorophore stability, and/or instrument stability" can affect results

• Never apply compensation matrices from previous experiments without new controls

• Use both compensation beads and single-stained cells as controls, particularly for surface markers with variable expression

What is the recommended protocol for using ERF095 antibody in Western blot applications?

For optimal Western blot results with ERF095 antibody, follow this validated protocol:

• Sample preparation: Prepare plant tissue extracts or recombinant protein containing the ERF095 target

• Gel electrophoresis: Use 12% SDS-PAGE for optimal resolution of the 16 kDa ERF095 protein

• Transfer: Blot to nitrocellulose (NC) membrane for 1 hour

• Blocking: Use 5% skim milk at room temperature or 4°C for 1 hour

• Primary antibody: Apply ERF095 antibody at 1:1000-1:2000 dilution overnight at 4°C

• Secondary antibody: Use goat anti-rabbit IgG H&L (HRP) at 1:10000 dilution

• Detection: Employ chemiluminescence substrate and capture images with CCD camera

How should ERF095 antibody be reconstituted and stored to maintain optimal activity?

Proper handling of ERF095 antibody significantly impacts experimental outcomes. To maintain antibody activity:

• Briefly centrifuge the vial prior to opening to prevent loss of lyophilized material adhering to the cap or sides

• Reconstitute lyophilized antibody with 150 μl of sterile water (or as recommended by manufacturer)

• For long-term storage, maintain at -20°C to -70°C for up to 6 months under sterile conditions after reconstitution

• For shorter storage periods, keep at 2-8°C for up to 1 month under sterile conditions

• Avoid repeated freeze-thaw cycles by preparing suitable aliquots upon reconstitution

How should researchers interpret multiple bands in Western blots using ERF095 antibody?

When multiple bands appear in Western blots with ERF095 antibody, systematic interpretation is required:

• Compare observed bands to the expected 16 kDa molecular weight of ERF095

• Consider potential isoforms or post-translational modifications that could alter migration patterns

• Evaluate if bands disappear in samples from ERF095 knockout lines (indicating specificity)

• Assess potential cross-reactivity with other ERF family members given the conserved DNA-binding domain

• Test an independent antibody targeting a different ERF095 epitope to confirm findings

What are common sources of background in ERF095 immunostaining and how can they be minimized?

Background issues in ERF095 immunostaining can compromise data interpretation. To minimize these problems:

• Optimize blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) and durations

• Titrate antibody concentration: Determine the minimum concentration providing specific signal through serial dilutions

• Include absorption controls: Pre-incubate antibody with recombinant ERF095 to confirm signal specificity

• Modify wash steps: Increase number, duration, or detergent concentration in wash buffers

• Evaluate tissue preparation: Optimize fixation methods, as overfixation can increase autofluorescence and background

How can ERF095 antibody be employed in chromatin immunoprecipitation (ChIP) studies?

ChIP applications with ERF095 antibody require specific considerations:

• Crosslinking optimization: Determine optimal formaldehyde concentration and crosslinking time for ERF095

• Sonication parameters: Establish conditions that generate 200-500 bp DNA fragments while preserving epitope integrity

• Antibody validation: Confirm antibody recognizes fixed/crosslinked forms of ERF095 before proceeding

• Controls: Include input samples, IgG controls, and positive controls targeting known DNA-binding proteins

• Target validation: Confirm ChIP results with orthogonal methods such as electrophoretic mobility shift assays

What approaches can quantify relative ERF095 expression levels across different experimental conditions?

For quantitative analysis of ERF095 expression:

• Western blot densitometry: Include calibration standards of recombinant ERF095 at multiple concentrations

• Quantitative immunofluorescence: Employ digital image analysis with appropriate normalization controls

• ELISA development: Consider sandwich ELISA using two antibodies targeting different ERF095 epitopes

• Comparative analysis: Always include reference/housekeeping proteins for normalization across samples

• Validation: Confirm protein-level changes with transcript-level analyses when possible

How can researchers predict and verify cross-species reactivity of ERF095 antibodies?

ERF transcription factors show varying degrees of conservation across plant species. To assess cross-reactivity:

• Perform sequence alignment of the immunogen peptide sequence across target species

• Test antibody reactivity against recombinant ERF095 proteins from different species

• Include species-specific positive and negative controls in all experiments

• Validate using genetic approaches (knockout/knockdown) when possible in non-model organisms

• Consider predicted reactivity information from manufacturers, which may indicate compatibility with mouse (99%), rat (99%), or bovine (99%) homologs

What methodological modifications are needed when applying ERF095 antibodies to different model systems?

When adapting ERF095 antibody protocols across species or systems:

• Optimize protein extraction methods based on tissue type and species-specific characteristics

• Adjust antibody concentrations based on target abundance in the specific model system

• Modify blocking reagents to address species-specific sources of background

• Consider species-appropriate positive controls (e.g., tissues known to express ERF095 homologs)

• Validate all protocol modifications through systematic testing and appropriate controls