EGR3 Antibody, Biotin conjugated

What is EGR3 and why is it a significant research target?

EGR3 (Early Growth Response 3) is a member of the early growth response (Egr) gene family of zinc finger transcription factors. It plays crucial roles in regulating genetic programs involved in cellular growth, differentiation, and function. EGR3 has particular significance as it:

• Functions as a master regulator of genes differentially expressed in neuropsychiatric conditions including schizophrenia, bipolar disorder, and Alzheimer's disease

• Regulates the expression of genes involved in inflammation, including those encoding various secreted cytokines and protease inhibitors

• Is involved in muscle spindle development as a probable transcription factor

Research has shown that EGR3 is one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated with antigen, indicating its potential role in allergic inflammation. The EGR3-HDAC6-IL-27 axis has been identified as a novel mediator in allergic inflammatory responses .

What are the structural characteristics of commercially available EGR3 antibodies?

Commercial EGR3 antibodies typically display the following characteristics:

Antibodies targeting EGR3 are commonly generated using recombinant fragments of the human EGR3 protein as immunogens, and they undergo antigen affinity purification to ensure specificity .

What protocols are recommended for biotinylating EGR3 antibodies for research use?

While direct information on EGR3 antibody biotinylation is not explicitly provided in the search results, standard antibody biotinylation methods can be adapted based on similar approaches. The recommended protocol involves:

• Buffer Exchange Preparation:

  • Dialyze antibody against 0.1 M sodium bicarbonate buffer (pH 8.3-8.5)

  • Ensure antibody concentration is 1-10 mg/mL for optimal conjugation

• Biotinylation Reaction:

  • Dissolve NHS-biotin or Sulfo-NHS-biotin in DMSO at 10 mg/mL

  • Add biotin reagent to antibody solution at a 20:1 molar ratio

  • Incubate at room temperature for 2 hours with gentle agitation

• Purification:

  • Remove unconjugated biotin using gel filtration or dialysis against PBS

  • Evaluate biotinylation efficiency using HABA assay or streptavidin binding assay

• Storage and Stability:

  • Store at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • Aliquot to avoid repeated freeze-thaw cycles

Alternatively, enzymatic biotinylation methods can be employed using a bacterial biotin ligase (BirA) recognition site (Avitag™) for site-specific conjugation, similar to the approach described for other proteins .

How can biotin-conjugated EGR3 antibodies be validated for specificity and sensitivity?

Proper validation of biotin-conjugated EGR3 antibodies requires a multi-faceted approach:

• Western Blot Validation:

  • Run appropriate positive controls (samples with known EGR3 expression)

  • Include negative controls (samples with knockout or knockdown of EGR3)

  • Confirm band at the expected molecular weight (43-50 kDa)

  • Compare with unconjugated antibody to ensure biotinylation hasn't affected specificity

• Immunoprecipitation Validation:

  • Perform IP using the biotin-conjugated antibody followed by detection with unconjugated anti-EGR3 antibody

  • Run reverse IP with unconjugated antibody followed by detection with streptavidin

  • Include appropriate controls such as IgG and no-antibody controls

• Blocking Experiments:

  • Pre-incubate the antibody with recombinant EGR3 protein

  • Compare signal intensity between blocked and unblocked antibody

• Cross-Reactivity Assessment:

  • Test antibody against related family members (EGR1, EGR2, EGR4)

  • Test across multiple species if cross-reactivity is claimed (human, mouse, rat)

What are recommended western blot protocols for biotin-conjugated EGR3 antibodies?

The optimal western blot protocol for biotin-conjugated EGR3 antibodies integrates standard techniques with specific considerations:

• Sample Preparation:

  • Use whole cell lysates or nuclear extracts from appropriate cell types (e.g., MCF7 cells, brain tissue)

  • Load 30-50 μg protein per lane for optimal detection

  • Use SDS-PAGE (7.5-10%) for optimal separation

• Transfer and Blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or 5% BSA in TBST

  • Critical step: If using streptavidin-HRP detection, ensure blocking agent does not contain endogenous biotin

• Detection Options:

  • Direct detection: Apply streptavidin-HRP (1:5000-1:10000) and visualize with ECL

  • Amplified detection: Use streptavidin-conjugated secondary reporter system

• Recommended Dilutions:

  • For biotin-conjugated EGR3 antibodies: 1:1000-1:4000 (optimize for each application)

  • Streptavidin-HRP: 1:5000-1:10000

• Controls:

  • Include positive control samples known to express EGR3

  • Include negative control without primary antibody

  • Consider using blocking peptide control

The expected molecular weight for EGR3 is 43 kDa, but observed weight may be up to 50 kDa depending on post-translational modifications .

How can non-specific binding be reduced when using biotin-conjugated EGR3 antibodies?

Non-specific binding is a common challenge when using biotin-conjugated antibodies. To minimize this issue:

• Optimizing Blocking Conditions:

  • Use freshly prepared blocking solutions

  • Consider alternative blocking agents (BSA, casein, commercial blockers)

  • For tissues/cells with high endogenous biotin, use avidin/biotin blocking kits prior to antibody application

• Buffer Optimization:

  • Increase salt concentration in wash buffers (150-500 mM NaCl)

  • Add 0.1-0.5% detergent (Tween-20, Triton X-100) to reduce hydrophobic interactions

  • Include 1-5% carrier proteins in dilution buffers

• Antibody Dilution Optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Typical dilution ranges from 1:1000 to 1:4000 for Western blotting

  • Excessive antibody concentration often increases background

• Pre-absorption Strategies:

  • Pre-incubate antibody with lysates from cells not expressing the target

  • For tissue immunostaining, consider pre-absorption with tissue powder

• Endogenous Biotin/Avidin Considerations:

  • Use commercial endogenous biotin blocking kits

  • Pre-block with unlabeled avidin followed by excess biotin

How does storage affect biotin-conjugated antibody performance, and what are the recommended storage conditions?

Proper storage is crucial for maintaining the activity and specificity of biotin-conjugated EGR3 antibodies:

Performance indicators that may suggest storage-related deterioration:

• Increased background in applications

• Reduced signal intensity

• Appearance of non-specific bands in Western blotting

• Decreased reproducibility between experiments

If deterioration is suspected, validation against a fresh aliquot or new lot should be performed .

How can biotin-conjugated EGR3 antibodies be utilized to study the EGR3-HDAC6-IL-27 axis in allergic inflammation?

Recent research has identified the EGR3-HDAC6-IL-27 axis as a critical mediator in allergic inflammation. Biotin-conjugated EGR3 antibodies can be strategically employed to investigate this pathway:

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • Use biotin-conjugated EGR3 antibodies to pull down EGR3-bound chromatin

  • Analyze binding to HDAC6 promoter sequences using PCR with specific primers:

    • HDAC6 promoter-1: 5′-TGGGCGGGCAAATGAAAAAG-3′ (sense) and 5′-GCCTACCGTTTAACCAGGCT-3′ (antisense)

    • HDAC6 promoter-2: 5′-GGATTCTGATCGAAAGGGGCA-3′ (sense) and 5′-TCCACTTCCCACATCCTTTCAT-3′ (antisense)

    • HDAC6 promoter-3: 5′-GGGTAGGGCAGGCCTAAGAA-3′ (sense) and 5′-CTAGATCGCAGCCTTCACCG-3′ (antisense)

• Co-Immunoprecipitation Studies:

  • Use in pull-down assays to isolate EGR3 and associated proteins

  • Analyze interactions between EGR3 and components of the NF-κB pathway

  • Identify protein complexes involved in HDAC6 regulation and IL-27 production

• In vivo Allergic Inflammation Models:

  • Apply in passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA) models

  • Track EGR3 expression and localization during allergic responses

  • Correlate with β-hexosaminidase activity as a measure of mast cell degranulation

• Multiplex Analysis with miR-182-5p:

  • Investigate how miR-182-5p (a negative regulator of EGR3) affects EGR3 localization

  • Combine with luciferase reporter assays using pGL3–3′-UTR-Egr3 constructs

What approaches can be used to employ biotin-conjugated EGR3 antibodies in kinase signaling pathway analysis?

Kinase signaling pathway analysis using biotin-conjugated EGR3 antibodies can be conducted through several sophisticated approaches:

• Kinase-Catalyzed Biotinylation (K-BMAPS) Integration:

  • Combine with ATP-biotin for kinase-catalyzed biotinylation

  • Use EGR3 antibodies to pull down EGR3 and analyze its phosphorylation state

  • Compare EGF-stimulated versus unstimulated samples to identify differential phosphorylation

  • Quantification protocol:

    • Normalize eluate signal intensity to input intensity

    • Express as percentage relative to EGF-stimulated sample

    • Perform statistical analysis using unpaired t-tests

• Multiplexed Pathway Analysis:

  • Study EGR3 in relation to other key signaling proteins:

    • EGFR and phospho-EGFR (Tyr1068)

    • ERK1/2 and phospho-ERK1/2 (Thr202/Tyr204)

    • AKT1 and phospho-AKT1 (Ser473)

  • Use streptavidin-based multicolor detection systems for simultaneous visualization

• Three-step Pretargeting Methods:

  • Adapt protocols similar to those using biotin/avidin ligand/anti-ligand binding pairs

  • Apply in sequential steps:

    1. Targeting moiety-ligand conjugate localization

    2. Anti-ligand binding and clearance

    3. Diagnostic agent binding

  • This approach can improve signal-to-noise ratio in complex signaling pathway analysis

How can researchers distinguish between EGR3 and other EGR family members when using biotin-conjugated antibodies?

Distinguishing between EGR family members requires careful experimental design and validation:

• Sequence-Based Selection Strategy:

  • Choose antibodies raised against unique regions of EGR3 not conserved in EGR1, EGR2, or EGR4

  • Target non-zinc finger domains which have greater sequence divergence

  • Avoid antibodies targeting the highly conserved zinc finger motifs

• Validation Experiments:

  • Cross-reactivity testing: Test antibody against recombinant EGR1, EGR2, EGR3, and EGR4

  • Knockout/knockdown controls: Use cells with specific EGR3 knockdown to confirm specificity

  • Peptide competition: Perform blocking experiments with specific EGR3 peptides

• Differential Expression Analysis:

  • Exploit known tissue-specific expression patterns of EGR family members

  • Brain tissue expresses multiple EGR members and requires careful validation

  • Compare expression patterns across tissues with known differential expression profiles

• Multi-antibody Approach:

  • Use multiple antibodies targeting different EGR3 epitopes

  • Concordant results increase confidence in specificity

  • Include specific antibodies against other EGR family members as controls

Researchers should also be aware of potential expression changes under different cellular conditions, as EGR3 can be rapidly induced by various stimuli, potentially complicating experimental interpretation .

What role might biotin-conjugated EGR3 antibodies play in studying neuropsychiatric disorders?

Biotin-conjugated EGR3 antibodies offer significant potential for neuropsychiatric research based on EGR3's established role as a master regulator of genes implicated in conditions such as schizophrenia, bipolar disorder, and Alzheimer's disease:

• Brain Region-Specific Expression Analysis:

  • Utilize biotin-conjugated antibodies for multiplex immunohistochemistry

  • Map EGR3 expression across different brain regions in neuropsychiatric models

  • Compare expression patterns between control and disease tissues

  • Advantage of biotin conjugation: Allows for signal amplification in tissues with low expression

• Single-Cell Resolution Studies:

  • Apply in flow cytometry and FACS sorting of neural cell populations

  • Combine with cell-type-specific markers for neuron subtypes, astrocytes, and microglia

  • Isolate EGR3-expressing cells for downstream transcriptomic or proteomic analysis

• Transcriptional Target Identification:

  • Use in ChIP-seq experiments to map EGR3 binding sites across the genome in neural tissues

  • Identify disease-relevant target genes regulated by EGR3

  • Compare binding patterns between healthy and disease states

  • Advantage of biotin-conjugation: Compatible with stringent wash conditions to reduce background

• Protein-Protein Interaction Networks:

  • Employ in pull-down assays to identify EGR3 interaction partners in neural cells

  • Characterize complexes involved in neuropsychiatric pathways

  • Combined with mass spectrometry for unbiased interaction screening

These approaches can help elucidate the molecular mechanisms by which EGR3 contributes to neuropsychiatric conditions, potentially identifying new therapeutic targets .

What are the latest methods for combining biotin-conjugated EGR3 antibodies with other molecular tools for multi-omics research?

Advanced multi-omics research can benefit from integrating biotin-conjugated EGR3 antibodies with other cutting-edge technologies:

• Spatial Transcriptomics Integration:

  • Combine immunostaining using biotin-conjugated EGR3 antibodies with spatial transcriptomics

  • Co-register protein localization with transcriptional profiles in tissue sections

  • Protocol adaptation:

    • Fix tissue sections using paraformaldehyde

    • Perform immunostaining with biotin-conjugated EGR3 antibody

    • Detect with streptavidin-fluorophore conjugate

    • Image tissue

    • Proceed with spatial transcriptomics workflow (e.g., Visium, Slide-seq)

    • Computational integration of protein and RNA data

• CUT&RUN and CUT&Tag Applications:

  • Adapt biotin-conjugated EGR3 antibodies for Cleavage Under Targets and Release Using Nuclease (CUT&RUN) or CUT&Tag methods

  • Advantage: Requires fewer cells than traditional ChIP-seq

  • Workflow modification:

    • Immobilize cells on ConA beads

    • Permeabilize cell membrane

    • Introduce biotin-conjugated EGR3 antibody

    • Add streptavidin-MNase or streptavidin-pA-Tn5

    • Controlled digestion and DNA recovery

    • Library preparation and sequencing

• Proximity Labeling Approaches:

  • Combine with BioID or APEX2 proximity labeling systems

  • Generate fusion constructs of EGR3 with biotin ligase (BirA*) or APEX2

  • Map the local interactome of EGR3 in different cellular contexts

  • Use biotin-conjugated EGR3 antibodies to validate identified interactions

• Single-Cell Proteogenomics:

  • Integrate with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing)

  • Develop oligonucleotide-conjugated EGR3 antibodies based on biotin-streptavidin linkage

  • Simultaneously profile EGR3 protein levels and transcriptome in single cells