ELK1 (Ab-383) Antibody

What is ELK1 and what cellular functions does it regulate?

ELK1 is a member of the ETS family of transcription factors and belongs to the ternary complex factor (TCF) subfamily. ELK1 functions as a nuclear target for the ras-raf-MAPK signaling cascade and forms a ternary complex by binding to the serum response factor and the serum response element in the c-fos proto-oncogene promoter . Recent research has demonstrated that ELK1:

• Acts as a transcriptional activator of downstream targets including proto-oncogenes

• Contributes to cell proliferation, migration, and invasion in cancer cells

• Regulates apoptosis pathways

• Functions as a coactivator with androgen receptor (AR) in certain cancer types

• Affects the expression of genes such as MMPs (matrix metalloproteinases) that play critical roles in cancer cell migration/invasion, angiogenesis, and metastasis

What is the specificity of ELK1 (Ab-383) Antibody and what epitope does it recognize?

ELK1 (Ab-383) Antibody is a rabbit polyclonal antibody that:

• Specifically targets the region around the phosphorylation site of Serine 383 in human ELK1

• Recognizes a peptide sequence spanning amino acids 381-385 (T-L-S-P-I) of human ELK1

• Detects endogenous levels of total ELK1 protein

• Shows cross-reactivity with human, mouse, and rat species

• Can be used to identify both phosphorylated and non-phosphorylated forms of ELK1, depending on the specific clone

What applications is ELK1 (Ab-383) Antibody validated for?

Based on validation data from multiple sources, ELK1 (Ab-383) Antibody has been successfully used in:

How should researchers optimize IHC protocols when using ELK1 (Ab-383) Antibody?

For optimal IHC results with ELK1 (Ab-383) Antibody:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections cut at 4-6 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Use 3-5% normal serum from the same species as the secondary antibody for 1 hour at room temperature.

• Primary antibody incubation: Apply ELK1 (Ab-383) Antibody at a 1:50-1:100 dilution in blocking buffer and incubate overnight at 4°C.

• Controls: Always include positive controls (breast carcinoma tissue shows strong expression) and negative controls (omit primary antibody or use peptide-blocked antibody) .

• Signal detection: For optimal visualization, use a biotin-streptavidin HRP system or polymer-based detection with DAB as the chromogen.

• Validation: Confirm specificity by showing reduced or absent staining when the antibody is pre-incubated with the immunizing peptide .

What are the critical parameters for Western blotting when using ELK1 (Ab-383) Antibody?

For successful Western blot analysis with ELK1 (Ab-383) Antibody:

• Sample preparation: Extract proteins using RIPA buffer containing protease and phosphatase inhibitors.

• Protein loading: Load 20-40 μg of total protein per lane.

• Gel percentage: Use 10% SDS-PAGE gels for optimal separation.

• Transfer conditions: Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody dilution: Dilute ELK1 (Ab-383) Antibody at 1:500-1:2500 in blocking buffer and incubate overnight at 4°C.

• Expected band size: ELK1 protein appears at approximately 44.9 kDa .

• Peptide competition: Run parallel samples with antibody pre-incubated with blocking peptide to confirm specificity.

How can researchers use ELK1 (Ab-383) Antibody to investigate androgen receptor signaling pathways?

ELK1 plays a significant role in androgen receptor (AR) signaling pathways, particularly in cancer biology. To investigate these interactions:

• Co-localization studies: Perform dual immunofluorescence with ELK1 (Ab-383) Antibody and anti-AR antibodies to examine co-localization in the nucleus after androgen treatment.

• Androgen stimulation experiments: Treat AR-positive cell lines (e.g., UMUC3, 647V-AR) with dihydrotestosterone (DHT) and/or AR antagonists like hydroxyflutamide (HF), then analyze ELK1 expression and nuclear translocation using this antibody .

• Transcriptional activity: Combine ELK1 (Ab-383) Antibody with ChIP assays to identify ELK1 binding to chromatin after androgen stimulation.

• Downstream target analysis: Use the antibody to correlate ELK1 activation with expression of downstream targets such as c-fos following androgen treatment .

• Knockdown studies: Perform ELK1 knockdown using shRNA in AR-positive cells and analyze the effects on androgen-induced cell proliferation and migration using this antibody as a validation tool .

Research has shown that DHT treatment significantly increases ELK1 expression and nuclear translocation in AR-positive bladder cancer cell lines, which can be antagonized by hydroxyflutamide .

What are the best approaches to study ELK1 phosphorylation status using this antibody?

To effectively study ELK1 phosphorylation:

• Dual antibody approach: Use both ELK1 (Ab-383) Antibody and phospho-specific ELK1 (Ser383) antibodies to distinguish between total and phosphorylated forms.

• Phosphatase treatment: Treat one set of samples with lambda phosphatase prior to immunoblotting to confirm phospho-specific detection.

• Stimulation experiments: Treat cells with MAPK pathway activators (e.g., EGF, PMA) to induce ELK1 phosphorylation and monitor changes in localization and activity.

• Inhibitor studies: Use MEK inhibitors (U0126, PD98059) to block the MAPK pathway and observe effects on ELK1 phosphorylation.

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions to track phosphorylation-dependent nuclear translocation of ELK1 .

Research has demonstrated that phosphorylated ELK1 (p-ELK1) expression is significantly elevated in urothelial neoplasms compared to non-neoplastic tissues, and positive p-ELK1 expression correlates with poor prognosis in bladder cancer patients .

How can ELK1 (Ab-383) Antibody be used to evaluate cancer progression biomarkers?

ELK1 has emerged as a potential biomarker for cancer progression. To utilize ELK1 (Ab-383) Antibody in cancer biomarker research:

• Tissue microarray analysis: Use the antibody at 1:50-1:100 dilution on tissue microarrays to correlate ELK1 expression with clinicopathological parameters across multiple patient samples.

• Prognostic correlation: As demonstrated in bladder cancer studies, use the antibody to assess relationships between ELK1/p-ELK1 expression and patient outcomes such as tumor recurrence, disease progression, and cancer-specific mortality .

• Multivariate analysis: Combine ELK1 staining with other biomarkers for Cox regression models to determine independent prognostic value.

• Quantitative assessment: Use digital image analysis for quantitative scoring of ELK1 immunostaining to establish objective cutoff values for positive expression.

Clinical studies have shown that patients with p-ELK1-positive non-muscle-invasive bladder tumors had significantly higher risks for tumor recurrence (p = 0.043), while those with muscle-invasive tumors had higher risks for disease progression (p = 0.045) and cancer-specific mortality (p = 0.008) .

What are the most common technical issues when using ELK1 (Ab-383) Antibody and how can they be resolved?

How should researchers validate the specificity of ELK1 (Ab-383) Antibody in their experimental system?

To ensure antibody specificity:

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (T-L-S-P-I sequence) before application to eliminate specific binding.

• Genetic knockdown/knockout controls: Compare antibody reactivity in wild-type versus ELK1 knockdown/knockout samples.

• Overexpression system: Test the antibody in cells overexpressing tagged ELK1 constructs.

• Multiple antibody comparison: Use other validated ELK1 antibodies targeting different epitopes and compare detection patterns.

• Cellular context validation: Test across different cell lines with varying ELK1 expression levels (e.g., UMUC3 with strong expression versus SVHUC with weak expression) .

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

How can ELK1 (Ab-383) Antibody be incorporated into multiplexed immunoassays?

For multiplexed analyses:

• Multi-color immunofluorescence:

  • Use ELK1 (Ab-383) Antibody with antibodies against related signaling molecules (e.g., AR, phospho-ERK)

  • Select compatible fluorophore conjugates for simultaneous detection

  • Perform sequential staining protocols if antibody species overlap exists

• Sequential chromogenic IHC:

  • Utilize ELK1 (Ab-383) Antibody in a sequential protocol with different chromogens

  • Employ antibody stripping or microwave treatment between staining rounds

  • Consider automated platforms for consistent results

• Mass cytometry (CyTOF):

  • Conjugate ELK1 (Ab-383) Antibody with rare earth metals for mass cytometry

  • Develop custom panels including ELK1 with other signaling proteins

  • Validate metal-conjugated antibodies against unconjugated versions

• Proximity ligation assay (PLA):

  • Combine ELK1 (Ab-383) Antibody with antibodies against potential interaction partners (e.g., AR)

  • Use this approach to visualize and quantify protein-protein interactions in situ

  • Optimize probe concentration and incubation conditions for maximum specificity

What experimental design is optimal for studying ELK1's role in cancer cell migration and invasion?

Based on published research with ELK1 (Ab-383) Antibody:

• Migration assays:

  • Perform scratch wound healing assays in ELK1-expressing versus ELK1-knockdown cells

  • Use time-lapse microscopy to track migration rates

  • Validate ELK1 expression/knockdown via Western blot with this antibody

  • Include androgen treatments to assess AR-ELK1 crosstalk effects on migration

• Invasion assays:

  • Employ transwell invasion assays with Matrigel-coated chambers

  • Quantify invasive cell populations after 24-48 hours

  • Correlate invasion capacity with ELK1 expression levels detected by this antibody

  • Analyze MMP expression and activity as downstream mediators of ELK1-driven invasion

• Molecular mechanism studies:

  • Assess MMP-2 and MMP-9 expression via qRT-PCR in ELK1-modulated cells

  • Perform gelatin zymography to measure MMP enzymatic activity

  • Use ChIP assays with ELK1 (Ab-383) Antibody to identify direct ELK1 binding to MMP promoters

  • Conduct rescue experiments by expressing MMP-2/9 in ELK1-knockdown cells

Research has demonstrated that ELK1 silencing significantly reduces cancer cell migration and invasion, correlating with decreased MMP-2 and MMP-9 expression and activity .

How can researchers integrate ELK1 (Ab-383) Antibody into studies examining the relationship between transcription factors and chromatin structure?

For chromatin-associated studies:

• Chromatin Immunoprecipitation (ChIP):

  • Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

  • Use ELK1 (Ab-383) Antibody at 5-10 μg per ChIP reaction

  • Include appropriate controls (IgG, input DNA, positive control regions)

  • Analyze ELK1 binding to known targets (e.g., c-fos promoter) and perform ChIP-seq for genome-wide binding

• ChIP-seq integration:

  • Compare ELK1 binding patterns with histone modification maps

  • Analyze co-occupancy with AR and other transcription factors

  • Correlate binding with gene expression changes after androgen treatment

  • Identify ELK1 binding motifs and potential novel targets

• Proximity-based methods:

  • Employ ChIP-MS approaches to identify ELK1 co-factors at chromatin

  • Use HiChIP with ELK1 (Ab-383) Antibody to examine long-range chromatin interactions

  • Integrate with ATAC-seq data to correlate ELK1 binding with chromatin accessibility

• Live-cell imaging:

  • Validate antibody's performance in immunofluorescence for ELK1 nuclear localization studies

  • Track ELK1 dynamics in response to signaling stimuli in real-time

What are the optimal storage conditions for maintaining ELK1 (Ab-383) Antibody activity?

For maximum stability and performance:

• Store at -20°C to -80°C for long-term storage

• Avoid repeated freeze-thaw cycles by making small working aliquots upon receipt

• For short-term storage (up to 2 weeks), refrigerate at 2-8°C

• The antibody is typically supplied in phosphate-buffered saline (pH 7.4) with 150mM NaCl, 0.02% sodium azide, and 50% glycerol for stability

• Expected shelf life is 12 months from date of receipt when stored properly

• When handling, keep on ice and return to storage promptly after use

• Follow safety precautions due to sodium azide content in buffer

ELK1 (Ab-383) Antibody Research Guide: Advanced Applications and Protocols

A comprehensive technical resource for optimizing ELK1-based investigations in cancer biology and signal transduction