ELK1 Antibody, HRP conjugated

What is ELK1 and what is its biological significance?

ELK1 is a transcription factor that belongs to the ETS family of proteins and binds to purine-rich DNA sequences. It plays a critical role in forming a ternary complex with Serum Response Factor (SRF) at the serum response element (SRE) found in the promoter regions of immediate early genes such as FOS and IER2. ELK1 is particularly important in the regulation of genes involved in cellular responses to extracellular stimuli. Following activation of JNK and MAPK signaling pathways, ELK1 induces target gene transcription, making it a critical component in signal transduction mechanisms and gene expression regulation . Phosphorylation of ELK1, especially at Ser383 and Ser389 by MAP kinases, enhances its interaction with co-activators such as p300, which plays a vital role in chromatin remodeling and gene activation .

What is an ELK1 Antibody, HRP conjugated, and what applications is it suitable for?

An ELK1 Antibody, HRP conjugated is an immunological reagent consisting of antibodies that specifically recognize ELK1 protein and are chemically linked to horseradish peroxidase (HRP) enzyme. This conjugation enables direct detection without requiring secondary antibodies. These antibodies are primarily used in applications such as Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and immunohistochemistry on paraffin-embedded tissues (IHC-P) . The direct HRP conjugation provides advantages in terms of reduced background signal and simplified experimental protocols. Commercial ELK1 antibodies demonstrate reactivity with human and mouse samples, with predicted reactivity for other species including rat, dog, cow, pig, horse, and rabbit based on sequence homology .

How should ELK1 Antibody, HRP conjugated be stored to maintain its activity?

ELK1 Antibody, HRP conjugated should be stored at -20°C, and it's advisable to aliquot the antibody into multiple vials to avoid repeated freeze-thaw cycles that can degrade antibody quality and reduce binding efficacy . The storage buffer typically contains 50% glycerol, which helps prevent freeze damage, along with stabilizers like 0.01M PBS or TBS (pH 7.4), 1% BSA, and 0.03% Proclin300 as a preservative . When working with the antibody, allow it to equilibrate to room temperature before opening the vial, and return unused portions to -20°C immediately after use. Proper storage is critical as deterioration can lead to inconsistent results in experimental applications.

What is the typical working dilution range for ELK1 Antibody, HRP conjugated in different applications?

The optimal working dilution varies depending on the specific application:

These ranges should be used as starting points for optimization in your specific experimental system . The optimal dilution will depend on multiple factors including the abundance of the target protein, the specific tissue or cell type being examined, and the detection method employed. It is recommended to perform a titration experiment when using the antibody for the first time in a particular application or with a new sample type.

How can I validate the specificity of an ELK1 Antibody, HRP conjugated?

Validating antibody specificity is essential for reliable research results. Several approaches can be employed:

• Positive and negative controls: Use cell lines or tissues known to express high levels of ELK1 as positive controls, and those with low or no expression as negative controls.

• Knockout/knockdown validation: Compare staining between wild-type samples and those where ELK1 has been knocked out or knocked down. A specific antibody should show reduced or absent signal in the knockout/knockdown samples .

• Peptide competition assay: Pre-incubate the antibody with an excess of the immunogen peptide before applying to your sample. Specific binding should be blocked, resulting in reduced or absent signal.

• Multiple antibody validation: Compare results using different antibodies targeting different epitopes of ELK1.

• Molecular weight verification: In Western blot applications, confirm that the detected band appears at the expected molecular weight of ELK1 (approximately 55 kDa) .

Remember that thorough validation is particularly crucial when studying phosphorylation-specific antibodies like anti-ELK1(Ser383), as these must demonstrate selectivity for the phosphorylated form over the non-phosphorylated form.

How can I use ELK1 Antibody, HRP conjugated to study the dynamics of ELK1 phosphorylation in response to MAP kinase signaling?

Studying ELK1 phosphorylation dynamics requires a methodical approach:

• Experimental design: Establish a time-course experiment exposing cells to stimuli known to activate MAP kinase pathways (e.g., thrombin, growth factors, stress inducers). Collect samples at multiple time points (e.g., 0, 5, 15, 30, 60 minutes) to capture the phosphorylation kinetics.

• Parallel antibody usage: Employ both phospho-specific ELK1 antibody (e.g., ELK1(Ser383)) and total ELK1 antibody to distinguish between changes in phosphorylation state versus changes in total protein level .

• Quantification methodology: Use densitometry analysis of Western blots to quantify the ratio of phosphorylated ELK1 to total ELK1 across your time points. This normalization is crucial for accurate interpretation of phosphorylation dynamics.

• Kinase inhibitor controls: Include conditions with specific MAP kinase inhibitors (JNK, ERK, p38) to confirm pathway specificity and identify which kinase predominantly phosphorylates ELK1 in your experimental context .

• Subcellular fractionation: Consider separating nuclear and cytoplasmic fractions to monitor potential translocation events associated with ELK1 activation, as phosphorylation may affect its nuclear localization.

This comprehensive approach will provide insights into both the temporal aspects of ELK1 phosphorylation and the specific signaling pathways involved in your experimental model.

What are the critical considerations when using phospho-specific ELK1 antibodies to study the interaction between ELK1 and p300 co-activator?

When investigating ELK1-p300 interactions using phospho-specific antibodies, several critical factors must be considered:

• Phosphorylation site specificity: Research shows that phosphorylation of ELK1 at Ser383 and Ser389 significantly enhances its interaction with p300 . Using antibodies specific to these phosphorylation sites is crucial for accurate assessment of the interaction dynamics.

• Experimental approaches:

  • Co-immunoprecipitation (Co-IP) using anti-ELK1 antibodies followed by p300 detection can reveal endogenous complex formation.

  • Reverse Co-IP using anti-p300 antibodies can confirm the interaction.

  • GST pull-down assays using recombinant ELK1 proteins (both wild-type and phospho-mimetic mutants) can help determine the direct effect of phosphorylation on p300 binding .

• Controls and validation:

  • Include phosphorylation-deficient mutants (e.g., S383A, S389A) as negative controls.

  • Use phosphatase treatment of samples to confirm phosphorylation-dependency.

  • Apply MAPK inhibitors to prevent ELK1 phosphorylation and observe effects on p300 interaction.

• Temporal considerations: Evidence suggests that ELK1 and p300 form a pre-assembled complex that becomes fully activated upon ELK1 phosphorylation . Design time-course experiments to capture both the pre-assembled state and the activation dynamics.

• Functional correlation: Correlate the observed ELK1-p300 interaction with functional outcomes such as histone acetyltransferase activity and target gene expression to establish biological relevance .

These considerations will help establish the relationship between ELK1 phosphorylation status and its functional interaction with the p300 co-activator in transcriptional regulation.

How can I distinguish between different phosphorylation states of ELK1 using available antibodies?

Distinguishing between different phosphorylation states of ELK1 requires a strategic approach:

• Site-specific phospho-antibodies: Utilize antibodies that specifically recognize ELK1 phosphorylated at distinct sites, such as Ser383, Ser389, or other relevant residues . These enable detection of specific phosphorylation events rather than general phosphorylation status.

• Combination analysis: Run parallel Western blots or immunoassays with different phospho-specific antibodies to create a phosphorylation profile. This approach can reveal whether certain sites are co-phosphorylated or independently regulated.

• Phosphatase treatment controls: Treat duplicate samples with lambda phosphatase to remove all phosphorylation modifications as a negative control for phospho-specific antibodies.

• Kinase-specific induction: Selectively activate different MAPK pathways (ERK, JNK, p38) using pathway-specific stimuli and inhibitors to determine which kinases phosphorylate which sites on ELK1.

• Mutational analysis: Use cells expressing ELK1 with serine-to-alanine mutations at specific phosphorylation sites to validate antibody specificity and to study the functional consequences of phosphorylation at individual sites.

• Mass spectrometry validation: For definitive phosphorylation site mapping, complement antibody-based approaches with mass spectrometry analysis of immunoprecipitated ELK1 to identify all phosphorylation sites present under different conditions.

This multi-faceted approach allows researchers to create a comprehensive map of ELK1 phosphorylation states and their functional implications in various cellular contexts.

What is the optimal protocol for using ELK1 Antibody, HRP conjugated in Western blot analysis?

The following protocol optimizes Western blot analysis with ELK1 Antibody, HRP conjugated:

Sample Preparation:

• Extract protein from cells or tissues using appropriate lysis buffer containing phosphatase and protease inhibitors to preserve phosphorylation status.

• Determine protein concentration using Bradford or BCA assay.

• Mix 20-50 μg of protein with loading buffer containing SDS and β-mercaptoethanol.

• Heat samples at 95°C for 5 minutes to denature proteins.

Gel Electrophoresis and Transfer:

• Resolve proteins on 10% SDS-PAGE gel (ELK1 has a molecular weight of approximately 55 kDa) .

• Transfer proteins to PVDF or nitrocellulose membrane using standard transfer conditions.

Antibody Incubation:

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• For phospho-specific detection, use 5% BSA in TBST as blocking agent rather than milk (milk contains phospho-proteins that can interfere).

• Dilute ELK1 Antibody, HRP conjugated in blocking buffer at 1:1000 dilution (optimize as needed within the 1:300-5000 range) .

• Incubate membrane with diluted antibody overnight at 4°C with gentle agitation.

• Wash membrane 3-5 times with TBST, 5 minutes each.

Detection:

• Apply ECL substrate directly to the membrane (no secondary antibody needed due to HRP conjugation).

• Expose to X-ray film or capture images using a digital imaging system.

• For quantitative analysis, use densitometry software to measure band intensity.

Controls:

• Include positive control (cell line known to express ELK1).

• Run a lane with molecular weight marker.

• Consider using GAPDH or β-actin as loading control .

This protocol should be optimized based on your specific experimental conditions and the particular ELK1 antibody being used.

How can I optimize ELK1 Antibody, HRP conjugated for immunohistochemistry on paraffin-embedded tissues?

Optimizing ELK1 Antibody, HRP conjugated for IHC-P requires systematic refinement:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours.

• Process and embed in paraffin following standard protocols.

• Section tissues at 4-6 μm thickness and mount on positively charged slides.

• Dry slides overnight at 37°C.

Antigen Retrieval Optimization:

• Test multiple antigen retrieval methods:

  • Citrate buffer (pH 6.0) for 20 minutes

  • EDTA buffer (pH 9.0) for 20 minutes

  • Tris-EDTA buffer (pH 9.0) for 20 minutes

• Use pressure cooker, microwave, or water bath methods to determine optimal retrieval conditions.

Blocking and Antibody Incubation:

• Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes.

• Block non-specific binding with 5% normal serum (from the same species as the secondary antibody would be, if not using direct HRP conjugate).

• Perform antibody titration experiment using dilutions from 1:200 to 1:400 .

• Incubate sections with antibody at 4°C overnight in a humidified chamber.

• Wash thoroughly with PBS or TBS buffer.

Detection and Counterstaining:

• Since the antibody is HRP-conjugated, apply DAB substrate directly.

• Monitor color development microscopically (typically 2-10 minutes).

• Counterstain with hematoxylin for nuclear visualization.

• Dehydrate through graded alcohols, clear in xylene, and mount with permanent mounting medium.

Controls and Validation:

• Include positive control tissue known to express ELK1.

• Include negative control by omitting primary antibody.

• For phospho-specific antibodies, include control slides treated with lambda phosphatase.

• Consider dual staining with total ELK1 antibody and phospho-specific antibody on serial sections to compare localization patterns.

This systematic approach will help establish optimal conditions for specific and sensitive detection of ELK1 or phospho-ELK1 in tissue sections.

What are the best practices for using ELK1 Antibody, HRP conjugated in ELISA applications?

Implementing best practices for ELISA with ELK1 Antibody, HRP conjugated ensures reliable results:

Direct ELISA Protocol:

• Coat 96-well plate with antigen (recombinant ELK1 protein or cell lysate) in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C.

• Wash wells 3 times with washing buffer (PBS with 0.05% Tween-20).

• Block non-specific binding with 5% BSA or 5% non-fat dry milk in PBS for 2 hours at room temperature.

• Wash wells 3 times with washing buffer.

• Prepare antibody dilution series (1:500-1:1000) in antibody dilution buffer (1% BSA in PBS-T) .

• Add diluted ELK1 Antibody, HRP conjugated to wells and incubate for 2 hours at room temperature.

• Wash wells 5 times with washing buffer.

• Add TMB substrate and incubate until color develops (typically 5-30 minutes).

• Stop reaction with 2N H₂SO₄ or equivalent stop solution.

• Read absorbance at 450 nm with reference wavelength at 630 nm.

Sandwich ELISA Approach:

• For detecting phosphorylated ELK1, consider a sandwich ELISA:

  • Coat plate with a capture antibody against total ELK1.

  • Add sample containing ELK1 protein.

  • Detect with phospho-specific ELK1 Antibody, HRP conjugated.

• This approach increases specificity for detecting phosphorylated forms within complex samples.

Critical Optimization Factors:

• Antibody concentration: Titrate to determine optimal working dilution.

• Sample preparation: For cell/tissue lysates, ensure complete lysis and determine optimal protein concentration.

• Incubation times and temperatures: Adjust based on signal intensity and background levels.

• Washing stringency: Increase number of washes if background is high.

Controls and Standards:

• Include a standard curve using recombinant ELK1 protein.

• Run phosphorylated and non-phosphorylated ELK1 standards for phospho-specific antibodies.

• Include blank wells (no antigen) and negative control wells (irrelevant protein).

• For phospho-specific detection, include samples treated with phosphatase as negative controls.

Following these methodological guidelines will optimize the sensitivity and specificity of ELK1 detection in ELISA applications.

What are common problems and solutions when using ELK1 Antibody, HRP conjugated in experimental applications?

Addressing common issues with ELK1 Antibody, HRP conjugated requires systematic troubleshooting:

Western Blot Issues:

IHC/ICC Issues:

ELISA Issues:

These troubleshooting strategies should be applied systematically while changing only one variable at a time to identify the specific cause of experimental issues.

How do phosphorylation-specific ELK1 antibodies differ from total ELK1 antibodies in research applications?

Understanding the distinct applications of phosphorylation-specific versus total ELK1 antibodies is crucial for experimental design:

Epitope Recognition and Specificity:

• Phospho-specific antibodies (e.g., ELK1(Ser383)) are raised against synthetic phosphopeptides containing the phosphorylated amino acid residue of interest . They recognize ELK1 only when phosphorylated at specific sites, enabling detection of active forms of the protein.

• Total ELK1 antibodies recognize epitopes independent of phosphorylation status, detecting all forms of ELK1 regardless of activation state .

Research Applications Comparison:

Methodological Considerations:

• Phospho-antibodies require careful sample handling with phosphatase inhibitors and often perform better with BSA (not milk) for blocking. They are particularly sensitive to dephosphorylation during sample preparation.

• Total antibodies are generally more robust to variations in sample preparation and provide reliable detection across different experimental conditions.

Complementary Usage:
The most informative experimental design often employs both antibody types in parallel to distinguish between changes in phosphorylation state versus changes in protein expression. This dual approach is particularly valuable in time-course experiments studying ELK1 activation dynamics following stimulation .

What experimental approaches can demonstrate the functional significance of ELK1 phosphorylation at Ser383 in transcriptional activation?

Demonstrating the functional significance of ELK1 phosphorylation at Ser383 requires a multi-faceted experimental approach:

1. Site-Directed Mutagenesis Studies:

• Generate phospho-mimetic (S383D or S383E) and phospho-deficient (S383A) ELK1 mutants.

• Compare their transcriptional activity using luciferase reporter assays with promoters containing serum response elements (SREs).

• Assess their ability to activate immediate early genes such as FOS and IER2 in cells lacking endogenous ELK1 expression .

2. Chromatin Immunoprecipitation (ChIP) Analysis:

• Use phospho-specific ELK1(Ser383) antibody to perform ChIP followed by qPCR or sequencing.

• Compare chromatin occupancy patterns at target gene promoters before and after stimulation with MAPK activators.

• Analyze histone acetylation status at these promoters to correlate ELK1 phosphorylation with chromatin remodeling .

3. Co-activator Interaction Studies:

• Perform co-immunoprecipitation experiments using wild-type ELK1 and S383A mutant to compare p300/CBP binding.

• Use GST pull-down assays with phosphorylated and non-phosphorylated recombinant ELK1 to assess direct interactions with co-activators.

• Analyze the histone acetyltransferase (HAT) activity of ELK1-associated complexes to determine if Ser383 phosphorylation enhances enzymatic activity .

4. Kinase Inhibitor Approach:

• Treat cells with specific MAPK pathway inhibitors (e.g., U0126 for MEK/ERK) prior to stimulation.

• Measure both ELK1 Ser383 phosphorylation and target gene expression.

• Correlate the degree of phosphorylation inhibition with transcriptional repression.

5. Live Cell Imaging:

• Generate fluorescent protein-tagged ELK1 constructs (wild-type and S383A).

• Use phospho-specific antibodies with immunofluorescence to track localization and activation dynamics in real-time following stimulation.

• Correlate nuclear accumulation patterns with transcriptional output.

6. Genomic Approaches:

• Perform RNA-seq comparing cells expressing wild-type versus S383A ELK1 mutant following MAPK pathway activation.

• Identify genes differentially regulated by phosphorylation at this specific site.

• Integrate with ChIP-seq data to create a comprehensive map of Ser383 phosphorylation-dependent gene regulation.

These complementary approaches collectively establish the mechanistic link between ELK1 phosphorylation at Ser383, co-activator recruitment, chromatin modification, and transcriptional activation of target genes .

How have ELK1 antibodies contributed to our understanding of immediate-early gene regulation in cellular stress responses?

ELK1 antibodies, particularly phospho-specific variants, have been instrumental in elucidating immediate-early gene regulation mechanisms:

• Pre-assembly Mechanism Discovery: Studies using ELK1 antibodies revealed that ELK1 forms pre-assembled complexes with coactivators like p300/CBP at enhancer elements before stimulation. This pre-assembly mechanism allows for rapid transcriptional activation following stress, which is critical for immediate-early response genes involved in stress responses . Without ELK1-specific antibodies, detecting these pre-formed complexes and their activation-dependent changes would have been impossible.

• Phosphorylation-Dependent Interaction Dynamics: Through co-immunoprecipitation experiments with phospho-specific ELK1 antibodies, researchers demonstrated that ELK1 phosphorylation at Ser383 and Ser389 by MAPKs enhances basal binding to p300 and establishes new interaction interfaces. These changes induce strong histone acetyltransferase activity in the ELK1-associated complex, facilitating chromatin remodeling needed for rapid gene activation .

• Temporal Regulation Insights: Time-course analyses using phospho-specific ELK1 antibodies have revealed the remarkably rapid kinetics of ELK1 phosphorylation following cellular stress. For example, studies have shown elevation of cIL-8 mRNA occurs within 7 minutes after thrombin stimulation, preceded by ELK1 phosphorylation . This temporal precision in signaling has been mapped through Western blot and immunofluorescence approaches using phospho-specific antibodies.

• Pathway Specificity in Stress Responses: Through selective usage of phospho-specific antibodies targeting different ELK1 phosphorylation sites, researchers have mapped which stress-activated pathways (JNK, p38, ERK) preferentially target specific residues. This has enabled the creation of phosphorylation signatures that correspond to distinct cellular stressors.

• Chromatin Dynamics Visualization: Combined application of ChIP techniques with phospho-specific ELK1 antibodies has allowed researchers to visualize the kinetics of chromatin association and modification at immediate-early gene promoters following stress, providing mechanistic insights into the speed of transcriptional responses.

These contributions have collectively transformed our understanding of immediate-early gene regulation from a simple on/off model to a sophisticated pre-assembly mechanism that enables extraordinarily rapid responses to cellular stress .

What methodological considerations are important when comparing results obtained with different ELK1 antibodies across studies?

When comparing studies using different ELK1 antibodies, researchers must consider several methodological factors to ensure valid interpretations:

• Epitope Specificity and Location:

  • Antibodies targeting different ELK1 epitopes may yield divergent results. N-terminal antibodies might detect all ELK1 isoforms, while C-terminal antibodies may miss truncated variants.

  • Phospho-specific antibodies recognize distinct phosphorylation sites (e.g., Ser383 vs. Ser389), which may be differentially regulated by upstream kinases .

  • Document the exact epitope recognized by each antibody when comparing studies.

• Antibody Format Differences:

  • Direct HRP-conjugated antibodies have different sensitivity profiles compared to unconjugated primary antibodies used with secondary detection systems .

  • Signal amplification methods vary between formats, potentially affecting detection thresholds.

  • Consider whether quantitative comparisons between studies using different formats are appropriate.

• Validation Standards Across Studies:

  • Older studies may have employed less rigorous validation methods than current standards require.

  • Evaluate whether antibodies were validated against knockout/knockdown controls, which represents the gold standard for specificity .

  • Consider whether phospho-antibodies were validated with phosphatase treatments or phospho-mimetic/deficient mutants.

• Sample Preparation Variations:

  • Differences in lysis buffers, fixation protocols, or antigen retrieval methods can significantly impact epitope accessibility.

  • Phosphorylation status is particularly sensitive to sample handling; inconsistent use of phosphatase inhibitors can cause major discrepancies.

  • Document and standardize sample preparation when possible for meta-analyses.

• Species Cross-Reactivity Considerations:

  • Antibodies may have different affinities for ELK1 from different species despite sequence conservation.

  • Some antibodies are validated for multiple species (human, mouse) while others have predicted reactivity based on homology .

  • Verify species-specific validation when comparing across model organisms.

• Detection Methods and Sensitivity:

  • Different visualization methods (ECL vs. fluorescence for Western blot; DAB vs. fluorescence for IHC) have different dynamic ranges.

  • Quantitative comparisons should account for linear range limitations of each detection method.

  • Digital imaging parameters (exposure time, gain settings) should be considered when comparing signal intensities.

• Experimental Context Variables:

  • Cell type-specific expression levels of ELK1 and its regulatory kinases may affect antibody performance.

  • Stimulation protocols (duration, concentration) influence phosphorylation status.

  • Create standardized positive controls when possible to normalize across experiments.

By systematically addressing these methodological variables, researchers can more accurately interpret apparent discrepancies between studies and develop more robust consensus models of ELK1 function in cellular signaling.