ELF3 Antibody, Biotin conjugated

What are the primary applications for ELF3 antibody, biotin conjugated, in cancer research?

Biotin-conjugated ELF3 antibodies are primarily used for detecting ELF3 protein expression in various cancer cell lines and tissue samples. The main applications include:

• Western blot analysis for protein detection and quantification

• Immunoprecipitation assays to study protein-protein interactions

• Immunohistochemistry for tissue localization studies

• ChIP (Chromatin Immunoprecipitation) assays to investigate ELF3 binding to promoter regions of target genes

Research has demonstrated that ELF3 antibodies can effectively detect ELF3 expression in prostate cancer cell lines (LNCaP), lung carcinoma cell lines (A549, PC-9), and mouse embryonic fibroblast cell lines (NIH-3T3) . The biotin conjugation significantly enhances detection sensitivity by allowing for streptavidin-based signal amplification, which is particularly valuable in samples with low ELF3 expression levels.

What are the recommended protocols for Western blot detection using biotin-conjugated ELF3 antibody?

For optimal Western blot detection of ELF3 using biotin-conjugated antibodies, researchers should follow these methodological steps:

• Prepare cell/tissue lysates under reducing conditions

• Separate proteins using SDS-PAGE (10-12% gel recommended)

• Transfer proteins to PVDF membrane

• Block membrane with appropriate blocking buffer (typically 5% non-fat milk or BSA)

• Incubate with biotin-conjugated ELF3 antibody (optimal dilution: 1:1000-1:2000)

• Detect using streptavidin-HRP conjugate (1:5000-1:10000 dilution)

• Visualize using enhanced chemiluminescence

Published data indicates that ELF3 is detected at approximately 42 kDa under these conditions . For optimal results, researchers should use Immunoblot Buffer Group 1 or equivalent to reduce background and non-specific binding.

How can I validate the specificity of biotin-conjugated ELF3 antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For biotin-conjugated ELF3 antibody, employ these validation approaches:

• Positive and negative control samples: Use cell lines known to express ELF3 (e.g., PC-3, A549) as positive controls and cell lines with low/no ELF3 expression as negative controls.

• siRNA knockdown validation: Perform siRNA-mediated knockdown of ELF3 using validated siRNAs (such as siELF3#1 and siELF3#2 targeting the coding region) . The signal from biotin-conjugated ELF3 antibody should significantly decrease in knockdown samples.

• Overexpression validation: Use ELF3-overexpressing cell models (such as TetOn-Flag-ELF3 systems) to confirm increased antibody signal.

• Competition assays: Pre-incubate the biotin-conjugated antibody with recombinant ELF3 protein before application to your samples. This should diminish specific signals.

• Peptide blocking: Use the immunizing peptide to block antibody binding, which should eliminate specific signals.

Studies have demonstrated that efficient ELF3 knockdown can be achieved using specific siRNAs, with western blot analysis showing a significant reduction in the 42 kDa band corresponding to ELF3 .

How can biotin-conjugated ELF3 antibody be utilized to investigate ELF3-AR interactions in prostate cancer research?

Biotin-conjugated ELF3 antibodies can be instrumental in studying the interaction between ELF3 and androgen receptor (AR) in prostate cancer through these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use biotin-conjugated ELF3 antibody to pull down ELF3 protein complexes, followed by western blot detection of AR. Research has shown that the ELF3-AR interaction is significantly enhanced in the presence of R1881 (synthetic androgen) .

• Proximity ligation assay (PLA): This technique allows visualization of protein-protein interactions in situ. Using biotin-conjugated ELF3 antibody alongside an AR-specific antibody enables detection of ELF3-AR proximity within cells.

• ChIP-reChIP assays: To investigate co-occupancy of ELF3 and AR at specific genomic loci, perform sequential ChIP with both antibodies.

• GST pull-down validation: Use recombinant GST-ELF3 and AR fragments to map interaction domains. Research indicates that both amino- and carboxy-terminal domains of AR can bind to ELF3 .

The interaction between ELF3 and AR has significant functional consequences, as ELF3 has been identified as a repressor of AR action in prostate cancer. This repressive function suggests that loss of ELF3 expression may contribute to prostate cancer pathogenesis through enhanced AR signaling .

What are the methodological considerations for using biotin-conjugated ELF3 antibody in ChIP experiments to identify ELF3 binding sites?

When using biotin-conjugated ELF3 antibodies for ChIP experiments, researchers should consider these methodological details:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature, as ELF3 binds DNA directly through its ETS domain.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp for optimal resolution.

• Antibody amount: Typically 2-5 μg of biotin-conjugated ELF3 antibody per ChIP reaction is sufficient. Determine optimal antibody concentration empirically for each experimental system.

• Streptavidin bead capture: Use streptavidin-coated magnetic beads for efficient capture of biotin-conjugated antibody-protein-DNA complexes.

• Positive controls: Include primers targeting known ELF3 binding sites. Research has identified ELF3 binding to promoter regions of multiple genes including EHF and TGIF1 .

• Data analysis: Calculate fold enrichment of ELF3 binding at target sites compared to IgG control immunoprecipitate. Published data shows significant enrichment (p<0.05) of ELF3 at promoter regions of master transcription factors in lung adenocarcinoma .

As demonstrated in lung adenocarcinoma models, ELF3 can bind to promoter regions of multiple genes, functioning within a regulatory network of transcription factors that drive cancer progression .

How can biotin-conjugated ELF3 antibody be used to investigate the role of ELF3 in BRCA1-associated breast cancer?

For investigating ELF3's role in BRCA1-associated breast cancer, biotin-conjugated ELF3 antibodies can be employed in these research approaches:

• Expression analysis: Use immunohistochemistry with biotin-conjugated ELF3 antibodies to analyze ELF3 expression patterns in BRCA1-mutated versus non-mutated breast cancer samples. Data from TCGA and METABRIC databases show significantly higher ELF3 expression in BRCA1-associated breast tumors than in non-BRCA1-associated breast tumors .

• Functional studies: Employ biotin-conjugated ELF3 antibodies in western blot analysis to monitor ELF3 expression changes following replication stress induction, which has been shown to upregulate ELF3 in BRCA1-associated contexts .

• Protein interaction network analysis: Use IP-mass spectrometry with biotin-conjugated ELF3 antibodies to identify protein interaction networks disrupted by high ELF3 expression in BRCA1-deficient cells.

• Target gene regulation: Perform ChIP-seq with biotin-conjugated ELF3 antibodies to identify genomic targets of ELF3 in BRCA1-deficient breast cancer models.

Research has identified several disrupted protein-protein interactions (PPIs) involving ELF3 in cancer contexts, including interactions with ERBB3, ETS1, and TIMP3, which may play roles in BRCA1-associated breast cancer progression .

What are common challenges when using biotin-conjugated ELF3 antibody in flow cytometry, and how can they be addressed?

When using biotin-conjugated ELF3 antibody for flow cytometry, researchers often encounter these challenges:

• Low signal intensity:

  • Problem: ELF3 is primarily a nuclear transcription factor, making detection challenging.

  • Solution: Ensure proper cell permeabilization using 0.1% Triton X-100 or saponin-based buffers. Use streptavidin conjugated to bright fluorophores (PE or APC) for signal amplification.

• High background signal:

  • Problem: Endogenous biotin can cause high background.

  • Solution: Block endogenous biotin using avidin/biotin blocking kits before incubation with biotin-conjugated ELF3 antibody.

• Specificity concerns:

  • Problem: Non-specific binding leading to false positives.

  • Solution: Include isotype controls and validate with siRNA knockdown controls as demonstrated in published studies .

• Fixation-induced epitope masking:

  • Problem: Some fixation methods may mask the ELF3 epitope.

  • Solution: Test multiple fixation protocols; methanol-based fixation often works better than formaldehyde for nuclear transcription factors.

Published research has successfully used ELF3 antibodies to detect expression differences between various cancer cell lines, suggesting that with proper optimization, flow cytometry can be effective for ELF3 detection .

What strategies should be employed to optimize immunohistochemistry (IHC) protocols using biotin-conjugated ELF3 antibody?

For optimal IHC results with biotin-conjugated ELF3 antibody, implement these methodological refinements:

• Antigen retrieval optimization:

  • Test multiple methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval)

  • Heat-induced epitope retrieval at 95-98°C for 20 minutes in citrate buffer often yields best results for nuclear transcription factors

• Endogenous biotin blocking:

  • Critical step: Use commercial avidin/biotin blocking kits before antibody application

  • Alternative: 0.1% avidin followed by 0.01% biotin incubation steps

• Signal amplification systems:

  • Standard: Streptavidin-HRP systems

  • Enhanced sensitivity: Tyramide signal amplification (TSA) for low-expressing samples

• Counterstaining considerations:

  • Light hematoxylin counterstaining (30 seconds) preserves nuclear ELF3 signal visibility

  • Avoid overstaining which can mask specific ELF3 nuclear staining

• Positive and negative controls:

  • Positive controls: Prostate cancer tissues (PC-3 xenografts), lung adenocarcinoma samples

  • Negative controls: Tissue sections incubated with isotype control antibodies

Research utilizing ELF3 immunohistochemistry has successfully differentiated expression patterns between tumor subtypes, particularly showing higher expression in lung adenocarcinoma compared to adjacent non-malignant tissue .

How does ELF3 expression vary across different cancer types, and what are the methodological considerations for detection?

ELF3 expression shows significant variation across cancer types, with important implications for biotin-conjugated ELF3 antibody detection strategies:

For methodological consistency across cancer types:

• Use standardized protein extraction protocols optimized for nuclear proteins

• Include appropriate positive controls (cell lines with known ELF3 expression)

• Consider tissue-specific fixation and permeabilization requirements

• Normalize expression to appropriate housekeeping proteins (GAPDH or β-actin)

How can biotin-conjugated ELF3 antibody be used to investigate ELF3's role as an oncogene in lung adenocarcinoma?

For investigating ELF3's oncogenic role in lung adenocarcinoma (LUAD), biotin-conjugated ELF3 antibodies can be employed in these methodological approaches:

Research has demonstrated that ELF3 overexpression significantly increases cell proliferation in human bronchial epithelial cells, suggesting its oncogenic potential in lung adenocarcinoma development .

What considerations should be made when using biotin-conjugated ELF3 antibody in multiplexed immunofluorescence assays?

When employing biotin-conjugated ELF3 antibody in multiplexed immunofluorescence assays, researchers should address these methodological considerations:

• Antibody panel design:

  • Carefully select compatible antibodies raised in different host species

  • For ELF3 interaction studies, include antibodies against known interactors (AR, ERBB3, ETS1)

  • When studying cancer contexts, include lineage markers relevant to cancer type (LUAD vs. LUSC markers)

• Signal separation optimization:

  • Use streptavidin conjugated to spectrally distinct fluorophores (Alexa Fluor 488, 555, 647)

  • Implement spectral unmixing for closely overlapping fluorophore emissions

  • Consider sequential detection rather than simultaneous for challenging multiplex combinations

• Controls for multiplexed assays:

  • Single antibody controls to confirm specificity and absence of crosstalk

  • Fluorescence minus one (FMO) controls to establish gating boundaries

  • Absorption controls to verify absence of secondary antibody cross-reactivity

• Image acquisition and analysis:

  • Use confocal microscopy for optimal spatial resolution of nuclear ELF3 signals

  • Employ automated image analysis software with nuclear segmentation capabilities

  • Quantify co-localization using established metrics (Pearson's correlation, Mander's overlap)

Research has successfully demonstrated ELF3 co-localization with AR in nuclei of LNCaP cells following R1881 treatment, highlighting the feasibility of multiplexed approaches to study ELF3 interactions .

How can biotin-conjugated ELF3 antibody be used in conjunction with single-cell technologies for heterogeneity analysis?

Integrating biotin-conjugated ELF3 antibody with single-cell technologies provides powerful approaches to analyze cellular heterogeneity:

• Single-cell mass cytometry (CyTOF):

  • Conjugate ELF3 antibody with biotin, followed by detection with metal-labeled streptavidin

  • Include markers for cell cycle phases, differentiation states, and lineage markers

  • Enables high-dimensional analysis of ELF3 expression in relation to multiple cellular states

• Single-cell Western blotting:

  • Use biotin-conjugated ELF3 antibody to detect ELF3 protein at single-cell resolution

  • Can reveal heterogeneity in ELF3 expression levels not detectable in bulk analysis

  • Particularly valuable for analyzing mixed cell populations in tumor samples

• Imaging mass cytometry (IMC):

  • Apply biotin-conjugated ELF3 antibody followed by metal-tagged streptavidin

  • Preserves spatial context while delivering single-cell resolution

  • Can identify rare ELF3-expressing cells within complex tissue architectures

• CITE-seq approaches:

  • Combine biotin-conjugated ELF3 antibody protein detection with transcriptome analysis

  • Correlate ELF3 protein levels with gene expression signatures at single-cell level

  • Particularly valuable for identifying cells with discordant mRNA/protein levels

These approaches can address important biological questions, such as the heterogeneity of ELF3 expression in luminal progenitor cells in BRCA1 mutation carriers, which have been identified as cells of origin for BRCA1-deficient breast cancers .

How can biotin-conjugated ELF3 antibody be used in combination with genetic manipulation techniques to study ELF3 function?

Biotin-conjugated ELF3 antibody can be effectively integrated with genetic manipulation approaches through these methodological strategies:

• CRISPR/Cas9 gene editing validation:

  • Use biotin-conjugated ELF3 antibody to confirm successful knockout/knockin

  • Western blot or immunofluorescence to verify complete protein loss in knockout models

  • Detect tagged ELF3 variants in knockin models

• siRNA/shRNA knockdown studies:

  • Validate knockdown efficiency using biotin-conjugated ELF3 antibody

  • Research has demonstrated successful ELF3 knockdown using siRNAs targeting coding regions

  • Compare effects of transient (siRNA) versus stable (shRNA) knockdown on ELF3 expression

• Conditional expression systems:

  • Monitor ELF3 expression in doxycycline-inducible systems like TetOn-Flag-ELF3

  • Research shows doxycycline-induced ELF3 expression effectively inhibits AR target gene expression

  • Western blot analysis can confirm dose-dependent induction of ELF3 expression

• Rescue experiments:

  • After ELF3 knockdown/knockout, reintroduce wild-type or mutant ELF3 variants

  • Use biotin-conjugated ELF3 antibody to confirm expression of rescue constructs

  • Distinguish between endogenous and exogenous ELF3 using epitope tags

Research has demonstrated that ELF3 overexpression inhibits prostate cancer cell growth in xenograft models, while knockdown increases androgen-dependent migration of prostate cancer cells , highlighting the utility of combining genetic manipulation with antibody-based detection.

What are the methodological approaches for using biotin-conjugated ELF3 antibody in prognostic studies of cancer patients?

For prognostic studies utilizing biotin-conjugated ELF3 antibodies, researchers should implement these methodological approaches:

Research demonstrates significant prognostic value of ELF3 expression in specific cancer subtypes, with particularly strong prognostic significance in Stage I LUAD patients (log-rank p = 1.00E-06) , indicating the potential clinical utility of ELF3 as a prognostic biomarker.

How can biotin-conjugated ELF3 antibody be utilized in translational research for targeted therapy development?

Biotin-conjugated ELF3 antibodies can support translational research for targeted therapy development through these methodological approaches:

• Target validation studies:

  • Use biotin-conjugated ELF3 antibody to confirm target expression in preclinical models

  • Validate on-target effects of ELF3-directed therapeutics via western blot

  • Monitor changes in ELF3 expression following therapeutic intervention

• Combination therapy research:

  • Evaluate ELF3 expression changes in response to standard therapies

  • Research shows ELF3 expression is affected by small-molecule inhibitors targeting SE-associated key targets (BRD4, EP300, CDK7)

  • Use biotin-conjugated ELF3 antibody to monitor expression in combination therapy studies

• Patient stratification biomarker development:

  • Standardize ELF3 detection protocols for potential companion diagnostic applications

  • Establish clinically relevant cutoff values for high versus low ELF3 expression

  • Correlate ELF3 expression with response to specific therapies

• Pharmacodynamic (PD) biomarker applications:

  • Use biotin-conjugated ELF3 antibody to monitor target engagement and modulation

  • Develop protocols for pre- and post-treatment biopsies to evaluate ELF3 expression changes

  • Correlate with clinical outcomes and drug efficacy

Research has demonstrated that inhibition of super-enhancer-associated targets reduces ELF3 expression and attenuates malignant progression in lung adenocarcinoma models , suggesting potential therapeutic approaches targeting ELF3 regulatory pathways.