EGR3 Antibody

What is EGR3 protein and what is its molecular weight?

EGR3 (Early Growth Response 3, also known as PILOT) is a zinc finger transcription factor involved in muscle spindle development, neuronal development, and immune regulation. The protein weighs approximately 43 kDa based on its calculated molecular weight (387 amino acids), though it may appear as a 50 kDa band in some experimental systems . EGR3 contains a highly conserved DNA-binding domain and belongs to the EGR family of immediate-early transcription factors that regulate critical genetic programs in cellular growth, differentiation, and function .

What types of EGR3 antibodies are commercially available and what are their key specifications?

Several types of EGR3 antibodies are available for research applications:

How does EGR3 protein structure relate to antibody epitope selection?

EGR3 protein has multiple functional domains, including a zinc finger DNA-binding domain and an N-terminal transcriptional activation domain. Research shows that antibodies targeting different regions detect distinct isoforms of EGR3. For example, antibodies directed against the N-terminal domain (αEgr3-NT) detect only the full-length isoform, while antibodies targeting internal epitopes (αEgr3-INT spanning amino acids 101-189) can detect both the full-length and truncated isoforms generated through alternative translation start sites . This structural understanding is critical when selecting antibodies for specific research applications, especially when investigating particular EGR3 isoforms.

What are the optimal conditions for using EGR3 antibodies in Western blot applications?

For optimal Western blot results with EGR3 antibodies:

• Sample preparation: Use 20-50 μg of protein lysate per lane. MCF7 cells, brain tissue lysates, and nuclear extracts provide reliable positive controls .

• Recommended dilutions:

  • Abcam antibody: 1/300-1/500 dilution

  • Cell Signaling antibody: 1/1000 dilution

  • Proteintech antibody: 1/1000-1/4000 dilution

• Detection system: HRP-conjugated anti-rabbit or anti-mouse IgG antibodies work effectively .

• Expected results: A primary band at approximately 43-50 kDa. Note that EGR3 can appear as multiple bands due to alternative translation start sites. The major band typically corresponds to the full-length isoform .

• SDS-PAGE conditions: 7.5-12% gels provide good resolution for EGR3 protein .

How can researchers validate the specificity of EGR3 antibodies?

Validating EGR3 antibody specificity is crucial for reliable experimental results. Multiple approaches include:

• Positive and negative controls: Use MCF7 cells (especially estrogen-treated) as positive controls, which express both major EGR3 isoforms. The larger molecular weight isoform increases with estrogen treatment .

• Comparison with recombinant protein: Test antibodies against recombinant EGR3 proteins. In one study, researchers transfected 293T cells with plasmids encoding different EGR3 isoforms (NM and BC) and confirmed antibody specificity via Western blot .

• Cross-validation with multiple antibodies: Use antibodies from different sources targeting distinct epitopes of EGR3. Consistent results across different antibodies increase confidence in specificity .

• Genetic knockouts: When possible, compare results with EGR3 knockout models. Egr3 knockout mice were generated by deleting the DNA binding domain and a 3.4-kb portion of the 3′ untranslated region of the Egr3 gene , providing definitive negative controls.

• Pre-absorption tests: Pre-incubate the antibody with its specific immunogen peptide to confirm that the resulting signal elimination is due to specific binding.

What are the key considerations for co-immunostaining experiments with EGR3 antibodies?

When designing co-immunostaining experiments with EGR3 antibodies:

• Fixation protocol: For optimal results, use 4% formaldehyde fixation, which preserves both protein structure and cellular morphology .

• Compatible co-staining markers:

  • α-tubulin for spindle structure visualization

  • γ-tubulin for MTOCs and spindle poles identification

• Avoiding cross-reactivity: When co-staining, ensure secondary antibodies do not cross-react. Use different host species for primary antibodies or employ directly conjugated primary antibodies.

• Subcellular localization controls: Include appropriate controls for subcellular compartments. For example, when studying EGR3 in nuclear vs. cytoplasmic locations, include known nuclear and cytoplasmic markers.

• Image acquisition: For proper co-localization analysis, use confocal microscopy with sequential scanning to avoid bleed-through between channels.

As demonstrated in one study, researchers successfully co-stained mouse oocytes with anti-EGR3 and anti-γ-tubulin antibodies to visualize the association between EGR3 and MTOCs, revealing that EGR3 accumulates near γ-tubulin-positive MTOCs .

How can researchers effectively study the multiple isoforms of EGR3 protein?

Research has identified multiple EGR3 isoforms generated through alternative translation start sites. To effectively study these variants:

• Isoform-specific detection: Use antibodies targeting different regions of EGR3. Antibodies directed against the N-terminal domain (αEgr3-NT) detect only the full-length isoform, while antibodies targeting internal epitopes (αEgr3-INT) can detect both full-length and truncated isoforms .

• Expression analysis: Studies show two major EGR3 mRNA transcripts (NM_018781.2 and BC103568) in various tissues. The larger isoform (NM) is predominantly translated in mouse oocytes and other tissues .

• Mutagenesis approaches: Point mutations at alternative translation start sites (Met residues) can help identify the specific start sites used for each isoform. Research has confirmed that Met106 serves as an alternative translation initiation site for a shorter EGR3 isoform .

• Functional studies: To determine functional differences between isoforms, express specific isoforms in appropriate cellular models. For example, researchers have used in vitro transcription/translation systems to synthesize different EGR3 isoforms and study their DNA binding properties using gel shift assays .

What approaches can be used to investigate EGR3's role in transcriptional regulation?

To study EGR3's function as a transcription factor:

• DNA binding assays: Use gel shift assays with the canonical EGR response element (ERE, 5′-GCGGGGGCG-3′). Studies show that EGR3 forms distinct DNA-binding complexes (α and β) corresponding to different isoforms .

• Reporter gene assays: Utilize ERE-containing luciferase reporter constructs (such as 1× ERE and 2× ERE) to measure EGR3-dependent transcriptional activity .

• Chromatin immunoprecipitation (ChIP): Identify genomic targets of EGR3 binding in vivo. EGR3 antibodies can be used to immunoprecipitate EGR3-bound chromatin fragments.

• Transcriptional profiling: Genome-wide expression profiling following EGR3 overexpression can identify regulated genes. One study found that genes associated with tissue remodeling and wound healing were prominently up-regulated by EGR3 .

• Pathway analysis: EGR3 is induced by transforming growth factor-β via canonical Smad3, suggesting its involvement in fibrotic responses . Investigating these signaling pathways can provide insight into EGR3's transcriptional role.

How can researchers address contradictory data regarding EGR3 localization in different cellular contexts?

EGR3 exhibits context-dependent localization patterns, which can lead to seemingly contradictory observations. To address these contradictions:

How do EGR3 antibodies perform across different tissue types and what are optimal sample preparation methods?

EGR3 expression varies across tissues, affecting antibody performance:

For optimal tissue-specific preparation:

• Brain tissue: Homogenize in RIPA buffer with protease inhibitors.

• Oocytes: Pool sufficient numbers (>100) to obtain adequate protein.

• Cell lines: For MCF7 cells, estrogen treatment (10 nM, 24h) enhances EGR3 expression.

• Nuclear extracts: Particularly useful for enriching transcription factors like EGR3.

What are the methodological considerations when investigating EGR3 in pathological conditions?

When studying EGR3 in disease contexts:

• Patient sample handling: For skin biopsy samples from patients with scleroderma, use 5-micron-thick paraffin-embedded or frozen sections and immunostain with primary antibody to Egr-3 followed by horseradish peroxidase–labeled secondary antibody .

• Quantification approaches: Score the pattern and expression of EGR3 staining in multiple randomly chosen high-power fields by a blinded observer to eliminate bias .

• Controls selection: Use age and sex-matched controls when studying disease samples. For scleroderma studies, researchers compared biopsy samples from patients with early-stage diffuse cutaneous scleroderma (n = 6) to healthy adults (n = 3) .

• Correlation with clinical parameters: EGR3 mRNA levels in scleroderma patients were found to correlate with the extent of skin involvement, suggesting its potential as a biomarker .

• Animal models: In mouse models of scleroderma, development of dermal fibrosis was accompanied by accumulation of Egr-3–positive myofibroblasts in the lesional tissue, providing a useful system to study EGR3's role in disease progression .

How can researchers optimize immunohistochemistry protocols for EGR3 detection in different tissue contexts?

For effective EGR3 immunohistochemistry:

• Tissue fixation: Use 4% paraformaldehyde for optimal antigen preservation.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) improves staining for most tissues.

• Antibody selection: For human samples, validated antibodies include those from Santa Cruz (A-7, sc-390967) and Cell Signaling (2559) .

• Protocol optimization by tissue type:

  • Brain tissue: Extended blocking (2-3 hours) with 5% BSA helps reduce background

  • Skin samples: For scleroderma studies, both paraffin-embedded and frozen sections yield reliable results

  • Ovarian tissue: To detect EGR3 in oocyte spindles, avoid over-fixation which can mask the epitope

• Visualization systems: For low-abundance expression, use amplification systems like tyramide signal amplification.

• Quantification: Use digital image analysis with appropriate thresholding to quantify EGR3 expression in different tissue compartments.

In scleroderma research, skin biopsy samples showed elevated Egr-3 levels in the dermis, demonstrating the utility of optimized IHC protocols for detecting disease-relevant expression patterns .

What are common problems encountered when using EGR3 antibodies and how can they be addressed?

Common challenges with EGR3 antibodies include:

• Multiple bands in Western blot:

  • Cause: Multiple isoforms due to alternative translation start sites

  • Solution: Use recombinant EGR3 isoforms as positive controls to identify specific bands

• Weak or no signal:

  • Cause: Low endogenous expression in many cell types

  • Solution: Use tissues with known high expression (brain, estrogen-treated MCF7 cells) as positive controls

  • Solution: Enrich samples by using nuclear extracts since EGR3 is primarily nuclear

• Non-specific binding:

  • Cause: Cross-reactivity with related EGR family proteins

  • Solution: Pre-absorb antibodies with recombinant related proteins

  • Solution: Validate with EGR3 knockout samples

• Inconsistent immunostaining:

  • Cause: Variability in fixation and permeabilization

  • Solution: Standardize fixation protocols (8% formaldehyde has been successful)

• Discrepancies between mRNA and protein levels:

  • Cause: Post-transcriptional regulation or protein stability issues

  • Solution: Compare RT-PCR and Western blot results from the same samples

What strategies can improve signal detection in samples with low EGR3 expression?

To enhance detection of low-abundance EGR3:

• Sample enrichment:

  • Use nuclear extraction for transcription factors like EGR3

  • Concentrate proteins by immunoprecipitation before Western blotting

• Signal amplification:

  • For Western blots: Use high-sensitivity ECL substrates

  • For IHC/IF: Employ tyramide signal amplification or polymer-based detection systems

• Optimized antibody conditions:

  • Extend primary antibody incubation (overnight at 4°C)

  • Test a range of antibody concentrations (typical successful range: 1-2 μg/ml)

• Inducing expression:

  • For cell lines, stimulate with appropriate factors (e.g., estrogen for MCF7 cells)

  • For neuronal tissues, seizure stimuli increase EGR3 expression

• Loading more protein:

  • For tissue samples with low expression, increase loading to 200 μg per lane

  • For oocytes, pool sufficient numbers (>100) to obtain adequate protein

How can researchers design experiments to distinguish between different EGR family members?

The EGR family includes several highly homologous transcription factors (EGR1, EGR2, EGR3, EGR4). To distinguish between them:

• Antibody selection:

  • Use antibodies targeting non-conserved regions outside the zinc finger domain

  • Validate specificity against recombinant proteins of all EGR family members

• Molecular approaches:

  • Design PCR primers for unique regions of each EGR transcript

  • Use nested PCR for low-abundance transcripts

• Expression pattern analysis:

  • Compare subcellular localization (EGR3 shows unique localization to spindles in oocytes)

  • Analyze tissue-specific expression patterns (EGR3 is the only EGR family member associated with MTOCs in oocytes)

• Functional discrimination:

  • Use gel shift assays with specific antibodies to identify which EGR protein is binding to DNA

  • Genome-wide expression profiling shows that <5% of fibroblast genes regulated by EGR1 or EGR2 are coregulated by EGR3, revealing substantial functional divergence

• Knockout validation:

  • Use tissue from specific EGR knockout models to confirm antibody specificity

  • Egr3 knockout mice lack muscle stretch receptors and show neural deficits not seen in other EGR knockouts