EGR2 Recombinant Monoclonal Antibody

What is EGR2 and what are its main functions in cellular biology?

Early growth response 2 (EGR2, also known as Krox-20) is a zinc finger-containing transcription factor that belongs to the early growth response family of proteins. These proteins function by binding to specific DNA regions to regulate the expression of target genes, thereby controlling various cellular processes . EGR2 is predominantly expressed in the thymus and nervous system, indicating its important role in these tissues' development and function. The protein contains DNA-binding zinc finger domains that enable its function as a transcriptional regulator.

EGR2 plays critical roles in both the immune and nervous systems. In the immune system, it is particularly important for the development of T cells and natural killer T (NKT) cells . Its expression is dynamically regulated during immune responses, with notable upregulation in T cells following T cell receptor (TCR) crosslinking. This suggests EGR2 serves as an important molecular switch during T cell activation processes. Research has also demonstrated that EGR2 can function as a negative regulator of T cell activation and may promote apoptosis under certain conditions, indicating its role in maintaining immune homeostasis .

How does EGR2 contribute to T cell development and immune regulation?

EGR2 serves as a critical transcriptional regulator in T cell development pathways, particularly in the thymus where it shows high expression levels . During T cell maturation, EGR2 helps orchestrate gene expression programs that guide proper cellular differentiation and functional specialization. Its expression patterns change dynamically throughout T cell development, suggesting stage-specific regulatory functions that help establish proper immune cell lineages.

What distinguishes EGR2 from other members of the EGR family?

The early growth response (EGR) family comprises several transcription factors (including EGR1, EGR2, EGR3, and EGR4) that share structural similarities but exhibit distinct biological functions. EGR2 possesses unique structural and functional properties that differentiate it from other family members, particularly in its tissue expression patterns and regulatory targets. While all EGR proteins contain zinc finger DNA-binding domains, subtle differences in these regions account for their distinct gene targeting profiles and biological functions.

The specificity of anti-EGR2 antibodies is particularly important for research applications, as cross-reactivity with other EGR family members could compromise experimental interpretations. The erongr2 monoclonal antibody has been specifically validated to recognize EGR2 without cross-reacting with closely related family members such as EGR3, as confirmed through immunoblot analysis . This specificity is critical when investigating EGR2-specific functions, especially in experimental systems where multiple EGR family members may be expressed simultaneously. Researchers should verify antibody specificity when studying EGR2 in various biological contexts, as the distinct functions of different EGR proteins could otherwise lead to confounding results and misinterpretations of experimental data.

What defines a recombinant monoclonal antibody and how does it differ from traditional monoclonal antibodies?

Recombinant monoclonal antibodies represent a technological advancement over traditional hybridoma-derived monoclonal antibodies. While traditional monoclonal antibodies are produced by immortalized hybridoma cell lines (created by fusing antibody-producing B cells with myeloma cells), recombinant monoclonal antibodies are generated through molecular cloning and recombinant DNA technology . This process typically involves isolating antibody gene sequences (heavy and light chains) from existing hybridomas or B cells, followed by cloning these sequences into expression vectors for production in host systems such as mammalian cells, bacteria, or yeast.

The recombinant approach offers several advantages over traditional hybridoma methods. For EGR2 antibody production, researchers can sequence existing hybridoma cell lines to obtain the primary sequences of antibodies targeting key epitopes . This process involves transcriptome sequencing to identify the antibody sequences, including both heavy and light chains, as well as native N-terminal signal peptides . Once identified, these sequences can be cloned into expression vectors for recombinant production, allowing for potentially unlimited production of antibodies with consistent properties. Additionally, recombinant technology enables antibody engineering, including modifications to improve specificity, affinity, or introduce desired functionalities like different detection tags or isotype switching.

What are the key characteristics of available EGR2 monoclonal antibodies?

The erongr2 monoclonal antibody represents a well-characterized tool for EGR2 detection in research applications. This antibody has been specifically developed to recognize mouse early growth response 2 (Egr2, Krox-20) with high specificity, without cross-reactivity to related proteins such as EGR3 . The antibody is classified as either IgG2a κ (as indicated in specification details) or other isotypes depending on the specific clone, which influences its binding properties and applications in different experimental systems.

These antibodies are available conjugated to various fluorophores, including phycoerythrin (PE) and allophycocyanin (APC), making them suitable for different flow cytometry applications . The PE-conjugated version has excitation ranges of 488-561 nm and emission at 578 nm, compatible with blue, green, and yellow-green lasers . The APC-conjugated version has excitation at 633-647 nm and emission at 660 nm, optimized for red laser detection systems . Both antibody preparations undergo 0.2 μm post-manufacturing filtration to ensure sterility and consistency, and they are typically supplied at a concentration of 0.2 mg/mL in PBS with 0.09% sodium azide (pH 7.2) . These well-defined spectral properties and physical characteristics enable researchers to incorporate EGR2 detection into multiparameter flow cytometry panels alongside other cellular markers.

What are the optimal conditions for using EGR2 monoclonal antibodies in flow cytometry?

Effective use of EGR2 monoclonal antibodies in flow cytometry requires careful optimization of several parameters to ensure specific and sensitive detection. The recommended antibody concentration for the erongr2 antibody is ≤0.25 μg per test, where a test is defined as the amount of antibody needed to stain a cell sample in a final volume of 100 μL . This concentration should be carefully titrated for each specific experimental system to determine the optimal signal-to-noise ratio. Cell numbers can range from 10^5 to 10^8 cells per test, though the specific number should be empirically determined based on the expected frequency of EGR2-expressing cells in the sample .

Since EGR2 is a transcription factor localized primarily in the nucleus, intracellular staining protocols are essential for its detection. The Foxp3/Transcription Factor Staining Buffer Set (Product #00-5523-00) has been validated for optimal results with these antibodies . This specialized buffer system provides appropriate fixation and permeabilization conditions that preserve cellular architecture while allowing antibody access to nuclear antigens. The staining procedure typically involves initial surface marker staining (if performing multiparameter analysis), followed by fixation/permeabilization, and finally intracellular antibody staining. Researchers should also consider including appropriate compensation controls when using fluorochrome-conjugated antibodies to correct for spectral overlap, particularly in multiparameter analyses combining EGR2 detection with other cellular markers.

How can researchers design optimal flow cytometry panels that include EGR2 detection?

Designing effective flow cytometry panels that incorporate EGR2 detection requires careful consideration of fluorochrome combinations, potential spectral overlap, and biological relevance of co-measured parameters. When selecting fluorochromes for EGR2 detection, researchers should consider the brightness hierarchy of available conjugates in relation to the expected expression level of EGR2. For instance, the PE-conjugated erongr2 antibody (excitation: 488-561 nm; emission: 578 nm) works well with blue, green, and yellow-green lasers, while the APC-conjugated version (excitation: 633-647 nm; emission: 660 nm) is optimized for red laser detection systems .

Since EGR2 expression shows dynamic changes following T cell stimulation, researchers should design panels that include relevant activation markers and functional readouts to correlate EGR2 expression with cellular states. For instance, including markers like CD69, CD25, or cytokine production can provide context for interpreting EGR2 expression patterns. When studying EGR2 in relation to T cell anergy, including phosphorylation markers of the ERK pathway can be informative, as EGR2 has been implicated in regulating ERK signaling during anergy induction . Panel design should also account for biological considerations such as the modest staining observed in subpopulations of T cells and B cells in freshly isolated spleen cells . This heterogeneous expression pattern necessitates inclusion of lineage markers in panels to properly identify and characterize EGR2-expressing subpopulations across different immune cell types.

What fixation and permeabilization methods are most effective for EGR2 detection?

Optimal detection of EGR2 requires specific fixation and permeabilization protocols that balance epitope preservation with sufficient cellular permeabilization to allow antibody access to nuclear transcription factors. The Foxp3/Transcription Factor Staining Buffer Set (Product #00-5523-00) has been specifically validated for use with erongr2 monoclonal antibodies and provides superior results compared to standard intracellular staining methods . This specialized buffer system contains optimized fixation components that preserve cellular architecture while providing adequate permeabilization for nuclear antigen detection.

How can researchers validate the specificity of EGR2 antibody staining?

Validating antibody specificity is crucial for generating reliable and interpretable data when studying EGR2 expression. Multiple complementary approaches should be employed to ensure the observed staining patterns truly represent EGR2. Positive controls should include samples where EGR2 expression is induced or upregulated, such as non-adherent mouse splenocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, which leads to measurable upregulation of EGR2 . These stimulated cells provide a biological system where EGR2 expression is dynamically regulated and can confirm antibody functionality.

Negative controls are equally important and should include isotype-matched control antibodies to assess non-specific binding. Additionally, researchers can utilize genetic approaches such as cells from EGR2-knockout models or cells where EGR2 has been deleted using Cre-recombinase systems to confirm staining specificity . The erongr2 monoclonal antibody has been specifically validated to not cross-react with the closely related EGR3 protein based on immunoblot analysis, which is an important specificity consideration . For further validation, researchers can perform blocking experiments using recombinant EGR2 protein (the antibody was raised against E. coli-derived protein, aa 1-319) to competitively inhibit specific staining. Correlation of protein detection with mRNA expression analysis (such as RT-PCR or RNA-seq) can provide additional confirmation of specificity by demonstrating concordance between transcriptional and translational expression patterns of EGR2.

How can EGR2 monoclonal antibodies contribute to understanding T cell anergy mechanisms?

EGR2 monoclonal antibodies offer valuable tools for investigating T cell anergy, a state of functional unresponsiveness that represents an important immune regulatory mechanism. Research has established EGR2 as a critical transcriptional regulator involved in T cell anergy induction, with deletion of EGR2 preventing anergy and restoring Ras/MAPK signaling in T cells . To study this process, researchers can design experiments using erongr2 antibodies to track changes in EGR2 expression during anergy induction and correlate these changes with functional outcomes.

A comprehensive experimental approach might include stimulating T cells under anergy-inducing conditions (such as TCR stimulation without costimulation) versus activation conditions (TCR plus costimulation), followed by intracellular staining for EGR2 using the validated erongr2 antibody . This approach allows researchers to correlate EGR2 expression levels with functional readouts such as IL-2 production, proliferation, and phosphorylation of signaling molecules like ERK . Flow cytometric analysis using the properly titrated erongr2 antibody (≤0.25 μg per test) enables quantitative assessment of EGR2 expression at the single-cell level . This can be particularly informative when combined with cell division tracking dyes and functional markers to correlate EGR2 expression with proliferative capacity and effector functions across heterogeneous T cell populations, providing mechanistic insights into how EGR2 regulates T cell responsiveness.

What experimental designs are most effective for investigating EGR2's relationship with TCR signaling pathways?

Investigating the relationship between EGR2 and TCR signaling pathways requires carefully designed experiments that capture the dynamic interplay between these components. A comprehensive experimental approach would combine time-course analyses of EGR2 expression following TCR stimulation with parallel assessments of signaling pathway activation. Researchers can stimulate T cells with anti-CD3/CD28 antibodies or PMA/ionomycin at various timepoints, then perform intracellular staining for both EGR2 (using erongr2 antibody) and phosphorylated signaling molecules such as pERK, which has been linked to EGR2 function in T cell anergy .

Flow cytometric analysis using properly validated erongr2 antibodies enables correlation of EGR2 expression with signaling pathway activation at the single-cell level . This approach can be enhanced by genetic manipulation strategies, such as EGR2 knockdown or conditional deletion systems using Cre-recombinase, to directly assess how altering EGR2 levels affects TCR signaling pathways . When combined with functional readouts such as cytokine production or proliferation assays, these experimental systems can provide mechanistic insights into how EGR2 influences T cell activation thresholds and responsiveness. Researchers should ensure their experimental design includes appropriate controls for antibody specificity, such as isotype controls and EGR2-deficient cells, to validate that the detected signals truly represent the biological relationships being investigated.

How can EGR2 antibodies be applied in developmental and neurological studies?

EGR2 plays significant roles in neurological development, making EGR2 monoclonal antibodies valuable tools for developmental neuroscience research. Given that EGR2 is predominantly expressed in the nervous system alongside its presence in the thymus , these antibodies can help elucidate the transcription factor's function in neuronal differentiation, myelination processes, and neural circuit formation. Researchers can apply immunohistochemistry or immunofluorescence techniques using validated erongr2 antibodies to visualize spatial and temporal expression patterns of EGR2 across developmental stages in the nervous system.

For developmental studies, experimental designs might include analyzing EGR2 expression at different embryonic and postnatal timepoints to correlate its expression with key developmental milestones. The specificity of the erongr2 monoclonal antibody for EGR2 without cross-reactivity to EGR3 is particularly important in neurological studies, as multiple EGR family members may be expressed in overlapping patterns in the nervous system . Flow cytometric analysis can be adapted for neural progenitor cells or isolated neuronal populations to quantify EGR2 expression during differentiation processes. When combined with functional studies using genetic manipulation of EGR2 expression (such as conditional knockout models), these approaches can provide mechanistic insights into how EGR2 regulates neural development and function, potentially revealing important links between immune and nervous system development that could have implications for neuroimmunological disorders.

What considerations are important when using EGR2 antibodies across different species models?

When applying EGR2 monoclonal antibodies across different species models, researchers must carefully consider epitope conservation and validate antibody reactivity in each target species. The erongr2 monoclonal antibody has been specifically validated for recognition of mouse EGR2 (Krox-20) , and its application to other species requires thorough validation. Epitope mapping data indicates that this antibody was raised against E. coli-derived protein spanning amino acids 1-319 of EGR2 , so researchers should analyze sequence conservation of this region across species of interest before attempting cross-species applications.

Cross-reactivity validation should include positive and negative controls specific to each species. For positive controls, researchers can use tissues or cells known to express EGR2 in the target species, ideally with confirmatory approaches such as RT-PCR or Western blotting to verify expression. Stimulation conditions known to induce EGR2, such as PMA/ionomycin treatment of lymphocytes , can provide additional biological validation across species. Negative controls should include tissues or cells known not to express EGR2, as well as samples from EGR2-knockout models if available in the species of interest. When validating for flow cytometry applications, titration experiments should be performed for each species to determine optimal antibody concentrations, which may differ from the recommended ≤0.25 μg per test established for mouse samples . Special attention should be paid to fixation and permeabilization conditions, which might require optimization for different cell types across species to ensure adequate antibody access to the nuclear EGR2 protein.

What are the most common causes of background staining when using EGR2 antibodies?

Background staining with EGR2 antibodies can arise from multiple sources, each requiring specific troubleshooting approaches. One common source is inadequate blocking before antibody incubation, which can lead to non-specific binding. Researchers should ensure their protocols include appropriate blocking steps with protein solutions (such as BSA or serum) matched to the host species of secondary reagents. Another frequent cause is over-fixation of samples, which can create autofluorescence or expose epitopes that lead to non-specific binding. The recommended Foxp3/Transcription Factor Staining Buffer Set has been optimized to minimize these issues, but fixation times should still be carefully controlled .

Fluorochrome selection can significantly impact background levels, with some fluorochromes exhibiting higher inherent background in certain cell types. The PE and APC conjugates of the erongr2 antibody offer different spectral properties that may perform differently depending on the cellular context and other panel components . Inadequate washing between steps can allow residual antibody to create background signal, so researchers should ensure thorough washing with appropriate buffers. For intracellular staining specifically, incomplete permeabilization can lead to antibody trapping and non-specific staining patterns. The specialized permeabilization buffer in the recommended Foxp3/Transcription Factor Staining Buffer Set helps mitigate this issue , but permeabilization conditions should be carefully monitored. Finally, high antibody concentrations can increase background staining, highlighting the importance of proper titration. While the recommended concentration is ≤0.25 μg per test, researchers should perform titration experiments to determine the optimal concentration for their specific experimental system .

How can researchers address inconsistent staining results with EGR2 antibodies?

Inconsistent staining results with EGR2 antibodies can stem from several factors that researchers should systematically address. Variability in sample preparation represents a common source of inconsistency, particularly when working with primary cells like splenocytes. Standardizing isolation procedures, minimizing processing time, and maintaining consistent cell viability can significantly improve reproducibility. Since EGR2 expression is dynamically regulated in response to stimuli such as TCR-crosslinking or PMA/ionomycin treatment , inconsistent stimulation conditions can lead to variable expression levels. Researchers should carefully control stimulation parameters including reagent concentrations, timing, and temperature.

The intracellular nature of EGR2 staining creates additional technical challenges that can contribute to inconsistency. Batch-to-batch variations in fixation/permeabilization efficiency can significantly impact nuclear antigen detection. Preparing larger volumes of fixation/permeabilization buffers from the recommended Foxp3/Transcription Factor Staining Buffer Set for multiple experiments can help maintain consistency . Antibody stability represents another potential variable, as fluorochrome-conjugated antibodies can degrade with repeated freeze-thaw cycles or extended storage at inappropriate temperatures. The erongr2 antibody preparations should be stored at 4°C in the dark and should never be frozen to maintain optimal performance . Finally, instrument variability in flow cytometers can contribute to inconsistent results, particularly when analyzing samples on different instruments or after cytometer maintenance. Regular quality control with standardized beads and consistent instrument settings can help minimize these variables and improve reproducibility across experiments.

What approaches are recommended for quantifying EGR2 expression in flow cytometry data?

Accurate quantification of EGR2 expression from flow cytometry data requires careful application of appropriate gating strategies and analytical approaches. Researchers should establish a standardized gating hierarchy that first identifies viable cells and removes doublets before proceeding to population-specific analyses. Since EGR2 exhibits differential expression across immune cell subsets, with modest staining observed in subpopulations of T cells and B cells in freshly isolated spleen cells , lineage-specific gating is essential to meaningfully interpret expression patterns. Within each lineage, EGR2-positive populations should be defined using appropriate controls, including fluorescence-minus-one (FMO) controls and isotype-matched antibodies to establish accurate positive/negative boundaries.

For quantitative analyses, researchers should consider multiple metrics beyond simple percent positive cells. Mean or median fluorescence intensity (MFI) provides information about expression levels within positive populations, which can reveal subtle changes in EGR2 expression following experimental manipulations. For stimulation experiments, such as those using PMA/ionomycin to induce EGR2 upregulation , fold-change in MFI relative to unstimulated controls offers meaningful quantification of expression dynamics. When analyzing heterogeneous populations, biaxial plots combining EGR2 with lineage markers or functional readouts can reveal correlations between EGR2 expression and cellular states. For more complex analyses, dimensionality reduction techniques such as tSNE or UMAP can help visualize EGR2 expression patterns across multiple parameters simultaneously. Statistical analyses should be appropriate to the experimental design, with paired tests for before/after comparisons and attention to appropriate normalization when comparing across experimental batches.

How should researchers interpret conflicting results between EGR2 protein detection and mRNA expression data?

Discrepancies between EGR2 protein detection using antibodies and mRNA expression data represent a common challenge that requires careful interpretation. These inconsistencies can arise from multiple biological and technical factors. Post-transcriptional regulatory mechanisms may cause temporal disconnects between mRNA production and protein accumulation. EGR2, as a transcription factor involved in dynamic cellular processes like T cell activation and anergy , may exhibit rapid transcriptional changes that are not immediately reflected at the protein level due to translation delays, protein stability differences, or post-translational modifications that affect epitope recognition by antibodies.

Technical factors can also contribute to apparent discrepancies. The sensitivity thresholds differ between techniques like RT-PCR or RNA-seq for mRNA detection and flow cytometry or Western blotting for protein detection. Additionally, antibody accessibility issues in intracellular staining may lead to false negatives if fixation and permeabilization conditions are suboptimal, even when the protein is present . When faced with conflicting data, researchers should consider validation through alternative methodologies. For mRNA, combining bulk and single-cell approaches can provide more comprehensive expression profiles. For protein detection, comparing results from flow cytometry, Western blotting, and immunofluorescence microscopy can help confirm expression patterns. Time-course experiments comparing mRNA and protein levels after stimulation with agents like PMA/ionomycin can help establish the temporal relationship between transcription and translation for EGR2 in specific cellular contexts, providing insights into the regulatory mechanisms controlling its expression and potentially explaining observed discrepancies.