ELK4 Antibody

Basic Research Questions

• What is ELK4 and why is it important in cellular function?

  ELK4 is a transcription factor belonging to the ETS family, also known as SAP-1 (SRF accessory protein 1). It functions by forming a ternary complex with serum response factor (SRF), requiring DNA-bound SRF for complex formation. ELK4 makes extensive DNA contacts to the 5' side of SRF but cannot bind DNA autonomously . With a molecular weight of approximately 47 kDa, ELK4 plays critical roles in regulating cell proliferation, cell cycle progression, and transcriptional activation/repression . Its importance extends to various physiological processes, including mast cell activation and proliferation, and has been implicated in several cancer types, making it a significant target for research .

• What applications can ELK4 antibodies be used for in laboratory research?

  ELK4 antibodies can be utilized in multiple research applications:

  • Western Blot (WB): Most commonly used for detecting ELK4 protein expression levels at the expected molecular weight of 47 kDa

  • Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ELK4

  • Immunocytochemistry (ICC) and Immunofluorescence (IF): For visualizing cellular localization

  • Immunohistochemistry (IHC): For detecting ELK4 in tissue sections

  • Chromatin Immunoprecipitation (ChIP): For studying ELK4-DNA interactions

  • Co-immunoprecipitation (Co-IP): For investigating protein-protein interactions involving ELK4

  When selecting an application, researchers should verify the antibody's validation status for their specific application to ensure reliable results .

• What is the difference between monoclonal and polyclonal ELK4 antibodies, and when should each be used?

  The choice between monoclonal and polyclonal ELK4 antibodies depends on experimental requirements:

  Polyclonal ELK4 antibodies (like those from Proteintech 14666-1-AP or Abcam ab86002) recognize multiple epitopes on the ELK4 protein, providing higher sensitivity but potentially lower specificity . These are advantageous for:

  • Initial protein detection

  • Applications requiring high signal strength

  • Detection of denatured proteins in Western blots

  Monoclonal ELK4 antibodies target a single epitope, offering higher specificity but potentially lower sensitivity. These are preferable for:

  • Experiments requiring consistent lot-to-lot reproducibility

  • Applications where cross-reactivity must be minimized

  • Specific domain recognition within ELK4

  For critical research, validation using multiple antibody types is recommended to confirm findings .

• What sample types can be used with ELK4 antibodies?

  ELK4 antibodies have been validated for use with various sample types:

  • Cell lines: Successfully detected in HeLa, Jurkat, MCF-7, C4-2B, PC3, CaSki, and other human cell lines

  • Tissue samples: Effective in human cervical cancer tissue, prostate cancer tissue, and mouse liver tissue

  • Primary cells: Functional in bone marrow-derived mast cells (BMMCs)

  When using a particular sample type, optimizing antibody dilution is crucial as recommended dilutions vary between applications (typically 1:500-1:2000 for Western blot) . Sample preparation protocols should include appropriate lysis buffers (e.g., containing NP-40, Triton X-100, EDTA, glycerol, and protease inhibitors for co-IP applications) .

Advanced Research Questions

• How can I optimize ELK4 antibody performance for chromatin immunoprecipitation (ChIP) experiments?

  For successful ChIP experiments with ELK4 antibodies:

  1. Antibody selection: Use ChIP-certified antibodies, such as the Atlas Antibodies anti-ELK4 (HPA028863) , which have been specifically validated for this application

  2. Crosslinking optimization:

     • For standard ChIP: Use 1% formaldehyde for 10 minutes at room temperature

     • For detecting transient interactions: Consider using dual crosslinking with DSG followed by formaldehyde

  3. Sonication parameters:

     • Optimize sonication to achieve DNA fragments of 200-500 bp

     • Use a bioanalyzer to confirm fragment size distribution

  4. Antibody amount and incubation conditions:

     • Typically use 2-5 μg of antibody per ChIP reaction

     • Incubate overnight at 4°C with rotation

     • Pre-clear lysates with protein Sepharose to reduce background

  5. Washing stringency:

     • Use multiple washes with buffer containing 100 nM NaCl, 200 mM Tris (pH 8.0), 0.5% NP-40, and protease inhibitors

  6. Controls:

     • Include IgG negative control

     • Use a positive control antibody (like anti-histone)

     • Include input chromatin samples

     • Consider known ELK4 targets like FBXO22 as positive control loci

  7. Validation: Validate ChIP efficiency with qPCR before proceeding to sequencing

• What experimental approaches can be used to study ELK4's role in cell cycle progression?

  Based on recent research, several methodological approaches can be employed:

  1. ELK4 knockdown/knockout models:

     • Generate stable ELK4 knockdown cell lines using shRNA (shELK4) or CRISPR-Cas9 technology

     • Verify knockdown efficiency via Western blot and qRT-PCR

  2. Cell proliferation assessment:

     • CCK-8/MTT assays to measure cell viability over time (typically days 1, 3, and 5)

     • EdU incorporation assays to directly measure DNA synthesis

     • Colony formation assays for long-term proliferation effects

  3. Cell cycle analysis:

     • Flow cytometry with propidium iodide staining to quantify cell distribution across G1, S, and G2/M phases

     • ModFit LT software for cell cycle distribution analysis

  4. Molecular mechanism investigation:

     • RNA-seq analysis comparing wild-type and ELK4-deficient cells to identify differentially expressed genes

     • RT-qPCR validation of cell cycle-related genes

     • ChIP-qPCR to identify direct ELK4 binding to cell cycle gene promoters

     • Luciferase reporter assays to confirm transcriptional regulation

  5. Rescue experiments:

     • Re-express ELK4 in knockout cells to confirm phenotype specificity

     • Mutational analysis of ELK4 functional domains to identify critical regions

  In cervical and prostate cancer models, ELK4 knockdown has been shown to block G1 to S phase progression, demonstrating its regulatory role in cell cycle control .

• How can I validate the specificity of my ELK4 antibody for immunoblotting?

  Comprehensive validation of ELK4 antibody specificity should include:

  1. Positive and negative controls:

     • Positive controls: Use cell lines with known ELK4 expression (HeLa, Jurkat, MCF-7)

     • Negative controls:

       • Primary antibody omission

       • ELK4 knockdown/knockout samples as negative controls

  2. Expected molecular weight confirmation:

     • Verify detection at the expected 47 kDa band

     • Be aware that post-translational modifications may cause slight variations

     • Note that tagged recombinant ELK4 may show higher molecular weights (e.g., ~61 kDa for a 14 kDa tag)

  3. Peptide competition assay:

     • Pre-incubate antibody with immunizing peptide

     • The specific band should disappear in Western blot

  4. Cross-validation with multiple antibodies:

     • Test multiple antibodies targeting different ELK4 epitopes

     • Compare results between polyclonal and monoclonal antibodies

  5. Tissue/cell-specific expression pattern:

     • Compare expression levels across tissues/cells with known ELK4 expression patterns

     • HPV-positive cervical cancer cells (HeLa, CaSki) show higher ELK4 expression than normal cervical cells

  6. Optimized protocols:

     • Use appropriate blocking (typically 5% non-fat milk or BSA)

     • Optimize antibody dilution (typically 1:500-1:2000)

     • Include appropriate positive controls in each experiment

• What are the critical considerations when using ELK4 antibodies for studying protein-protein interactions?

  When investigating ELK4's interactions with other proteins:

  1. Co-immunoprecipitation (Co-IP) protocol optimization:

     • Lysis buffer selection: Use buffers containing 170 mM NaCl, 50 mM Tris (pH 8.0), 0.5% NP-40, 1% Triton X-100, 1 mM EDTA, 5% glycerol, and protease inhibitors

     • Pre-clearing step: Incubate lysates with protein Sepharose for 1 hour at 4°C to reduce non-specific binding

     • Antibody incubation: Allow overnight binding at 4°C

     • Washing stringency: Multiple washes with buffer containing 100 nM NaCl, 200 mM Tris (pH 8.0), 0.5% NP-40, and protease inhibitors

  2. Validated interaction partners:

     • MITF: ELK4 interacts with MITF to regulate cytokine/chemokine gene expression

     • SIRT6: ELK4 complexes with SIRT6 to regulate degranulation-related genes

     • SRF: ELK4 forms ternary complexes with SRF for transcriptional regulation

  3. Reciprocal Co-IP verification:

     • Confirm interactions by immunoprecipitating with antibodies against both ELK4 and the partner protein

     • For example, when studying ELK4-MITF interaction, perform Co-IP with both anti-ELK4 and anti-MITF antibodies

  4. Controls and validation:

     • IgG control: Include isotype-matched IgG as negative control

     • Input samples: Include input lysate control (typically 5-10%)

     • Functional validation: Support protein interactions with functional assays (e.g., reporter assays, ChIP-seq, etc.)

  5. Alternative approaches:

     • Proximity ligation assay (PLA) for in situ detection of protein interactions

     • FRET or BRET for studying dynamic interactions

     • Mass spectrometry for identifying novel interaction partners

• How do different ELK4 antibodies perform in detecting post-translational modifications?

  Detection of ELK4 post-translational modifications (PTMs) requires specialized approaches:

  1. Phosphorylation-specific antibodies:

     • ELK4 contains conserved consensus phosphorylation sites for MAP kinases in its C-box region

     • Phospho-specific antibodies against these sites can be used to monitor ELK4 activation status

     • When using phospho-specific antibodies, include both phosphatase inhibitors in lysis buffers and dephosphorylation controls

  2. PTM-sensitive applications:

     • Phos-tag SDS-PAGE can resolve phosphorylated from non-phosphorylated ELK4

     • 2D-gel electrophoresis may separate differently modified ELK4 forms

     • IP followed by mass spectrometry for comprehensive PTM mapping

  3. Functional correlation:

     • Correlate phosphorylation status with transcriptional activity using reporter assays

     • Compare wild-type ELK4 with phospho-mimetic or phospho-deficient mutants

  4. Application-specific considerations:

     • For Western blot: Use fresh samples and phosphatase inhibitors

     • For IF/IHC: Optimization of fixation methods is critical as some may mask PTM epitopes

  5. Methodological challenges:

     • PTM-specific antibodies often require extensive validation

     • Signal may be transient or present at low levels

     • Consider enrichment steps (IP) before detection

• What are the best strategies for quantifying ELK4 expression levels in disease models?

  For accurate quantification of ELK4 in disease models:

  1. RNA level quantification:

     • RT-qPCR using validated ELK4 primers and appropriate reference genes

     • RNA-seq for genome-wide expression comparison

     • In HPV-positive cervical cancer, ELK4 mRNA levels are significantly elevated compared to normal cervical tissue

  2. Protein level quantification:

     • Western blot with densitometry analysis

       • Include loading controls (β-actin, GAPDH, etc.)

       • Use standard curves with recombinant ELK4 for absolute quantification

     • ELISA for high-throughput quantification across multiple samples

  3. Tissue analysis:

     • Immunohistochemistry (IHC) with semi-quantitative scoring systems

       • Score staining intensity as negative (0), weak (1), moderate (2), or strong (3)

       • Use digital pathology software for objective quantification

     • Tissue microarrays for comparing multiple patient samples

  4. Disease-specific considerations:

     • Prostate cancer: ELK4 expression correlates with disease progression and can be regulated by βKlotho

     • Cervical cancer: Higher ELK4 expression in HPV-positive samples compared to HPV-negative samples

     • Consider correlating ELK4 levels with clinical parameters (e.g., Gleason score, PSA levels, metastasis status)

  5. Controls and normalizations:

     • Include normal adjacent tissue controls

     • Use matched patient samples where possible

     • Normalize to appropriate housekeeping genes/proteins

     • Consider cell-type-specific markers for heterogeneous samples

• How can I design experiments to study ELK4's role in transcriptional regulation?

  To investigate ELK4's function as a transcription factor:

  1. Identification of ELK4 target genes:

     • ChIP-seq to map genome-wide ELK4 binding sites

     • RNA-seq comparing wild-type and ELK4-deficient cells to identify differentially expressed genes

     • Integrate ChIP-seq and RNA-seq data to identify direct targets

     • Known targets include FBXO22, cell cycle genes, and cytokines/chemokines (Hdc, Ccl3, Ccl4)

  2. Promoter analysis:

     • Luciferase reporter assays with wild-type and mutated promoters

       • For example, ELK4 binding to the FBXO22 promoter has been validated using this approach

     • EMSA (Electrophoretic Mobility Shift Assay) to confirm direct DNA binding

     • ChIP-qPCR for targeted validation of binding to specific promoters

  3. Functional mechanisms:

     • Co-IP to identify transcriptional cofactors interacting with ELK4

       • Known partners include MITF for cytokine/chemokine regulation and SIRT6 for degranulation regulation

     • Knockdown/overexpression of ELK4 combined with gene-specific qRT-PCR

     • Mutation analysis of ELK4 functional domains

  4. Context-dependent regulation:

     • Compare ELK4 function across different cell types

     • Study ELK4 activity under different stimulation conditions

       • For instance, in mast cells, ELK4 expression is downregulated upon activation

  5. Advanced technologies:

     • CUT&RUN or CUT&Tag as alternatives to ChIP-seq

     • HiChIP to connect ELK4 binding with chromatin architecture

     • Single-cell approaches to address cellular heterogeneity

• What are the key technical challenges when using ELK4 antibodies across different applications and how can they be addressed?

  Common technical challenges with ELK4 antibodies include:

  1. Cross-reactivity concerns:

     • Issue: ELK4 belongs to the ETS family which has high sequence homology among members

     • Solution:

       • Use antibodies raised against unique regions of ELK4

       • Validate specificity using ELK4 knockout/knockdown controls

       • Consider peptide competition assays to confirm specificity

  2. Application-specific optimization:

     • Western Blot:

       • Challenge: Variable band intensity or multiple bands

       • Solution: Optimize blocking (5% milk or BSA), antibody dilution (1:500-1:2000), and exposure time

     • Immunofluorescence/IHC:

       • Challenge: High background or weak specific signal

       • Solution: Test different fixation methods, antigen retrieval techniques, and antibody dilutions (1:50-200 for IF/IHC)

     • ChIP:

       • Challenge: Low enrichment or high background

       • Solution: Optimize crosslinking, sonication conditions, and use ChIP-certified antibodies

  3. Sample preparation issues:

     • Problem: Degradation or modification loss during processing

     • Solution: Use fresh samples, appropriate protease/phosphatase inhibitors, and optimize lysis buffers for specific applications

  4. Quantification challenges:

     • Problem: Semi-quantitative nature of many detection methods

     • Solution:

       • Use standard curves with recombinant proteins

       • Include multiple technical and biological replicates

       • Apply appropriate statistical analysis

  5. Reproducibility between antibody lots:

     • Problem: Lot-to-lot variation, especially with polyclonal antibodies

     • Solution:

       • Purchase larger quantities of a single lot for long-term projects

       • Validate each new lot against previous lots

       • Consider monoclonal antibodies for critical applications