EGR3 Antibody, FITC conjugated

What is EGR3 and what are its biological functions?

EGR3 is a member of the early growth response transcription factor family of C2H2 zinc finger proteins, which includes EGR1, EGR2, and EGR4. EGR3 functions as an immediate early protein, with expression rapidly upregulated in response to a wide range of extracellular stimuli . It plays crucial roles in several biological processes, including:

• Muscle spindle development

• Brain response to stress or injury

• Transcriptional regulation of genes involved in cellular growth, differentiation, and survival

• Modulating immune response regulation, with rapid expression in T cells after activation

EGR3 primarily localizes to the nucleus where it functions as a transcriptional regulator, allowing it to respond swiftly to extracellular signals and modulate gene expression in real-time .

What does FITC conjugation mean in the context of EGR3 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation refers to the chemical linkage of the FITC fluorophore directly to an antibody molecule. This conjugation enables:

• Direct visualization of the target protein without requiring secondary antibodies

• Single-step immunofluorescence detection procedures

• Emission of green fluorescence when excited with light around 495nm wavelength

The conjugation process attaches FITC to amine groups on the antibody while preserving the antibody's specific binding properties. FITC-conjugated antibodies are particularly useful for applications requiring direct detection of target proteins in immunofluorescence and flow cytometry .

What research applications are most suitable for FITC-conjugated EGR3 antibodies?

FITC-conjugated EGR3 antibodies are particularly valuable for:

• Immunofluorescence (IF) microscopy of fixed cells and tissues

• Western blotting when used with appropriate secondary detection systems

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Studies examining EGR3 expression dynamics in response to stimuli like TGF-β

• Co-localization studies with other nuclear or cytoplasmic proteins

• Chromatin immunoprecipitation (ChIP) experiments to identify EGR3 binding sites on target genes

What is the recommended protocol for immunofluorescence using FITC-conjugated EGR3 antibodies?

A standard protocol for immunofluorescence with FITC-conjugated EGR3 antibodies includes:

Cell Preparation:

• Grow cells on coverslips or chamber slides

• Fix cells with an appropriate fixative (methanol is often effective)

• Wash cells 2-3 times with PBS

Immunostaining:

• Add 2mL of blocking solution (PBS containing 10% fetal bovine serum) and incubate for 20 minutes at room temperature

• Remove blocking solution and add 1mL of PBS/10% FBS containing FITC-conjugated EGR3 antibody (recommended dilution 1:500)

• Incubate for 1 hour at room temperature in the dark

• Wash cells 2× for 5 minutes with PBS

Visualization:

• Mount slides with appropriate mounting medium

• Observe cells with a fluorescence microscope equipped with a FITC filter

How should FITC-conjugated EGR3 antibodies be stored to maintain activity?

Proper storage is crucial for maintaining antibody activity:

• Store in light-protected vials or covered with a light-protecting material (e.g., aluminum foil)

• For short-term storage, keep at 4°C (stable for at least 12 months)

• For long-term storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freezing and thawing as this will compromise both enzyme activity and antibody binding

• Protect from continuous exposure to light, which causes gradual loss of fluorescence

What controls should be included when using FITC-conjugated EGR3 antibodies?

Proper experimental controls are essential:

• Negative control: Cells stained with isotype-matched irrelevant IgG conjugated with FITC at the same concentration

• Positive control: Cells known to express EGR3 (e.g., stimulated T cells or fibroblasts treated with TGF-β)

• Autofluorescence control: Unstained cells to assess natural cellular fluorescence

• Blocking peptide control: Pre-incubation with specific blocking peptides to confirm antibody specificity

• Secondary antibody-only control: When using unconjugated primary antibodies with FITC-conjugated secondary antibodies

How can background fluorescence be minimized when using FITC-conjugated antibodies?

Common sources of background and solutions include:

"If no signal or high background occurs, try alternative fixation methods. Poor fixation can mask epitopes while too much antibody causes excessive non-specific binding."

How do different EGR3 antibodies compare in terms of binding specificity and applications?

Different EGR3 antibodies target various regions of the protein, affecting their applications:

"EGR3 antibodies can be used to detect the expression patterns in response to various stimuli, as EGR3 is swiftly upregulated following extracellular signals" .

How can I validate the specificity of FITC-conjugated EGR3 antibodies?

Validation methods include:

• Western blot analysis: Confirm a single band at the expected molecular weight (approximately 43 kDa)

• Pre-absorption test: Pre-incubate antibody with immunizing peptide; specific staining should be eliminated

• siRNA knockdown: Signal should be reduced in cells with EGR3 knockdown

• Testing across multiple cell types: Compare staining patterns in cells with known EGR3 expression

• Chromatin immunoprecipitation (ChIP): For transcription factors like EGR3, validate binding to known target sequences

"This is a rabbit polyclonal antibody against EGR3. It was validated on Western Blot using a cell lysate as a positive control" .

How can I use FITC-conjugated EGR3 antibodies to study its role in disease models?

EGR3 has been implicated in several disease processes, making FITC-conjugated antibodies valuable for:

• Cancer research: EGR3 levels in tumor tissues can be assessed via immunofluorescence to study correlation with prognosis

• Fibrosis models: EGR3-positive myofibroblasts can be identified in fibrotic tissues, as EGR3 is induced by TGF-β in fibroblasts

• Neurological studies: EGR3 expression changes in brain regions following stress or injury can be visualized

• Immunological research: EGR3 dynamics in T cell activation can be tracked

"In a mouse model of scleroderma, development of dermal fibrosis was accompanied by accumulation of Egr-3–positive myofibroblasts in the lesional tissue. Moreover, skin biopsy samples from patients with scleroderma showed elevated Egr-3 levels in the dermis" .

What are the considerations for detecting nuclear versus cytoplasmic EGR3?

While EGR3 is primarily a nuclear protein, cytoplasmic localization has been reported:

• Nuclear detection: Standard fixation protocols are usually sufficient; counterstain with DAPI to confirm nuclear localization

• Cytoplasmic detection: "The exact functional role of cytoplasmic EGR3 remains elusive" but may be related to "cell division and cytoskeleton organisation"

• Differential localization: May require careful optimization of fixation and permeabilization protocols

• Co-localization studies: Can help determine if cytoplasmic EGR3 associates with specific cellular structures

For optimal visualization of both compartments, "different fixation methods can significantly impact staining results; test multiple fixation methods with your specific cell type" .

What might cause weak or no signal when using FITC-conjugated EGR3 antibodies?

How does EGR3 expression change temporally in response to stimuli?

Understanding temporal dynamics is crucial for experimental design:

• TGF-β stimulates EGR3 mRNA expression in a time- and dose-dependent manner

• Peak mRNA expression typically occurs around 60 minutes post-stimulation, followed by decline to near basal levels by 24 hours

• Protein levels show a maximal increase (>3-fold) at approximately 4 hours, followed by gradual decline

• For optimal detection of induced EGR3, cells should be fixed at appropriate time points (1-4 hours post-stimulation for peak expression)

"TGF-β stimulated Egr-3 mRNA expression in a time- and dose-dependent manner. A maximal fivefold increase was seen at 60 minutes, followed by decline to near basal levels by 24 hours" .

How can I optimize dual staining with FITC-conjugated EGR3 antibodies and other fluorophores?

For multi-color immunofluorescence:

• Choose compatible fluorophores: Select secondary fluorophores with minimal spectral overlap with FITC (e.g., Cy3, Alexa Fluor 594)

• Sequential staining: Apply FITC-conjugated EGR3 antibody first, followed by other primary/secondary antibody pairs

• Careful washing: Include additional washing steps between antibody applications

• Compensation controls: Prepare single-stained samples for each fluorophore to allow for spectral compensation

• Image acquisition: Capture FITC channel first to minimize photobleaching, as "FITC is more susceptible to photobleaching than many other fluorophores"

"For co-localization studies, select a second primary antibody raised in a different host species than the EGR3 antibody to avoid cross-reactivity."

How is EGR3 involved in disease pathogenesis and what research approaches can explore this?

EGR3 has been implicated in multiple disease processes:

• Cancer progression: "High nuclear levels of EGR3 remained significantly associated with poor patient survival in MGMT-methylated patients"

• Fibrotic disorders: "EGR3 mRNA levels correlated with the extent of skin involvement" in scleroderma patients

• Chemotherapy resistance: "EGR3 may have a protective function against chemotherapy" in some cancer types

• Neurological disorders: EGR3 is "upregulated in several regions of the brain in response to stress or injury"

Research approaches include comparative expression studies, colocalization with disease markers, genetic manipulation of EGR3 levels, and correlation with patient outcomes.

What are the advantages and limitations of FITC-conjugated antibodies compared to other detection methods?

"Continuous exposure to light will cause the FITC-conjugated antibody to gradually lose its fluorescence" .