ELP4 Antibody, HRP conjugated

What is ELP4 and what cellular functions does it perform?

ELP4 (Elongator complex protein 4) is a critical component of the RNA polymerase II elongator complex, which functions as a histone acetyltransferase within the RNA polymerase II holoenzyme. The protein plays essential roles in transcriptional elongation and is involved in chromatin remodeling processes. Specifically, ELP4 participates in the acetylation of histones H3 and likely H4, facilitating chromatin accessibility during transcription . More recent research has expanded our understanding, revealing that ELP4 is also required for multiple tRNA modifications, including mcm5U (5-methoxycarbonylmethyl uridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), and ncm5U . This dual functionality in both histone modification and tRNA processing positions ELP4 as a multifunctional protein involved in diverse cellular processes related to gene expression regulation.

How does HRP conjugation benefit the detection of ELP4?

Horseradish peroxidase (HRP) conjugation provides several significant advantages for ELP4 detection in research applications:

• Enhanced sensitivity: HRP enables enzymatic signal amplification, which substantially increases detection sensitivity compared to unconjugated primary antibodies that require secondary detection.

• Streamlined protocols: Direct HRP conjugation eliminates the secondary antibody incubation step, reducing experiment time, potential cross-reactivity issues, and background signal.

• Greater compatibility: HRP-conjugated antibodies work effectively across multiple detection methods, including Western blotting, ELISA, and immunohistochemistry, providing versatility for researchers employing different techniques .

• Quantitative analysis: The enzymatic reaction produces a colorimetric or chemiluminescent signal proportional to antigen concentration, allowing for more accurate quantification.

When working with low-abundance proteins like ELP4, which may be present in limited amounts within the elongator complex, this signal amplification capability becomes particularly valuable for detecting physiologically relevant expression levels.

What are the differences between polyclonal and recombinant (monoclonal) ELP4 antibodies?

The key differences between polyclonal and recombinant/monoclonal ELP4 antibodies impact experimental design and outcomes:

Both antibody types are suitable for Western blot, immunohistochemistry (IHC-P and IHC-F) , but researchers should select based on their specific experimental needs, with polyclonal options offering broader epitope recognition and monoclonal versions providing higher specificity.

What subcellular localizations of ELP4 should researchers expect to detect?

According to the search results, ELP4 demonstrates dual localization within both the cytoplasm and nucleus . This distribution pattern reflects the protein's multifunctional role:

• Nuclear localization: Within the nucleus, ELP4 functions as part of the elongator complex associated with RNA polymerase II, participating in transcriptional elongation and histone acetylation processes. The nuclear presence enables direct interaction with chromatin and the transcriptional machinery.

• Cytoplasmic localization: In the cytoplasm, ELP4 participates in tRNA modification activities that affect translation efficiency and accuracy.

When designing experimental controls and interpreting immunostaining results, researchers should anticipate this dual-compartment distribution pattern. Validation can include subcellular fractionation followed by Western blotting to confirm the presence of ELP4 in both compartments. Immunocytochemistry or immunofluorescence experiments should employ proper nuclear counterstains (e.g., DAPI) to verify the nuclear/cytoplasmic distribution pattern.

Which applications are most suitable for ELP4 antibody, HRP conjugated?

Based on the manufacturer's validated applications, ELP4 antibody with HRP conjugation is optimized for the following techniques:

• Western Blotting (WB): Highly recommended as the primary application with dilution ranges of 1:300-5000 , allowing for specific detection of ELP4 protein in cell and tissue lysates.

• Enzyme-Linked Immunosorbent Assay (ELISA): Effective at dilutions of 1:500-1000 , providing quantitative measurement of ELP4 in solution.

• Immunohistochemistry - Paraffin-embedded sections (IHC-P): Reliable for tissue localization studies at dilutions of 1:200-400 , enabling visualization of ELP4 in preserved tissue architecture.

• Immunohistochemistry - Frozen sections (IHC-F): Suitable for detection in frozen tissue sections at dilutions of 1:100-500 , offering an alternative approach for tissues sensitive to paraffin embedding.

When determining the optimal application for your research, consider that WB provides information about protein size and potential modifications, while immunohistochemistry techniques reveal spatial distribution within tissues. ELISA offers quantitative measurement capabilities but lacks spatial information. The choice should align with your specific research question regarding ELP4 function or expression.

How should Western Blot protocols be optimized when using ELP4 antibody, HRP conjugated?

To optimize Western blot protocols specifically for ELP4 antibody (HRP conjugated), researchers should implement the following methodological refinements:

• Sample preparation:

  • Include protease inhibitors to prevent degradation of ELP4

  • Consider phosphatase inhibitors if investigating potential post-translational modifications

  • Optimize lysis buffers with 1% BSA similar to the antibody storage buffer

• Gel electrophoresis parameters:

  • Use 8-10% acrylamide gels for optimal separation, as ELP4 protein's molecular weight falls within the range effectively resolved by these percentages

  • Ensure sufficient running time for proper separation

• Transfer conditions:

  • Implement wet transfer for 60-90 minutes at controlled temperature

  • Use PVDF membranes which have shown superior results with ELP4 detection, as demonstrated in multiple antibody validation protocols

• Blocking and antibody incubation:

  • Block with 1-5% BSA in TBS with 0.1% Tween-20 (compatible with the antibody formulation)

  • Start with a mid-range dilution (1:1000) for HRP-conjugated ELP4 antibody , then optimize

  • Incubate at 4°C overnight for optimal binding

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Begin with shorter exposure times (30 seconds) and increase as needed

  • Consider using signal enhancers for low-abundance samples

• Controls:

  • Include both positive controls (cell lines known to express ELP4) and negative controls

  • A loading control such as GAPDH or β-actin should be used for normalization

This protocol optimization will enhance the specificity and sensitivity of ELP4 detection, particularly important given its dual subcellular localization and potential post-translational modifications.

What dilution factors are recommended for different experimental applications?

Based on the manufacturer's specifications, the following dilution ranges are recommended for optimal results with ELP4 antibody, HRP conjugated:

It's important to note that these recommendations serve as starting points. Researchers should perform titration experiments to determine optimal concentrations for their specific experimental conditions, sample types, and detection methods. When optimizing dilutions, consider:

• Signal-to-noise ratio: Balance between specific signal and background

• Sample type: Different tissues/cells may require adjusted dilutions

• Detection method: Chemiluminescence vs. colorimetric detection

• Storage time: Antibodies stored for extended periods may require adjustment of dilution factors

Antibody titration experiments, using a dilution series across the recommended range, will help establish the optimal working concentration for each specific research application.

What are the best practices for immunohistochemistry using ELP4 antibody, HRP conjugated?

For optimal immunohistochemical detection of ELP4 using HRP-conjugated antibodies, researchers should follow these best practices:

• Tissue preparation:

  • For paraffin-embedded sections (IHC-P): Use 4-6 μm thick sections on positively charged slides

  • For frozen sections (IHC-F): Cut 8-10 μm sections and fix appropriately (acetone fixation for 10 minutes at -20°C works well for many antigens)

• Antigen retrieval (particularly important for IHC-P):

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended

  • Pressure cooker or microwave-based methods can enhance retrieval efficiency

• Blocking steps:

  • Block endogenous peroxidase activity with 0.3% H₂O₂ in methanol for 30 minutes

  • Block nonspecific binding with 1-5% BSA in PBS, which aligns with the antibody formulation buffer

• Primary antibody application:

  • Apply ELP4 antibody at dilutions of 1:200-400 for paraffin sections or 1:100-500 for frozen sections

  • Incubate overnight at 4°C in a humidified chamber for optimal binding

• Detection system:

  • Since the antibody is already HRP-conjugated, proceed directly to chromogenic detection

  • DAB (3,3'-diaminobenzidine) substrate is recommended for visualization, as demonstrated in reference protocols

  • Control incubation time (typically 2-10 minutes) while monitoring under a microscope to avoid overdevelopment

• Counterstaining and mounting:

  • Counterstain with hematoxylin for nuclear visualization (30-60 seconds)

  • Use permanent mounting medium for long-term preservation

• Controls:

  • Include tissue sections known to express ELP4 as positive controls

  • Use isotype control antibodies or omit primary antibody as negative controls

  • Consider dual staining with nuclear markers to confirm the expected subcellular localization

Following these methodological guidelines will help ensure specific and reproducible ELP4 detection in tissue samples while minimizing artifacts and background staining.

How can researchers troubleshoot weak or absent signals in ELP4 detection?

When encountering weak or absent signals in ELP4 detection experiments, consider the following systematic troubleshooting approach:

• Antibody-related factors:

  • Verify antibody viability: HRP activity can diminish over time or with improper storage

  • Check for excessive freeze-thaw cycles which can degrade both antibody and conjugated HRP

  • Confirm you're using the appropriate dilution range (1:300-5000 for WB; 1:200-400 for IHC-P)

  • Consider testing different lots or alternative anti-ELP4 antibodies

• Sample preparation issues:

  • Ensure complete protein denaturation for Western blots

  • Verify protein extraction efficiency from nuclear fractions, as ELP4 has dual localization

  • Check protein degradation by examining other proteins in the same sample

  • Consider using different lysis buffers optimized for nuclear proteins

• Detection system optimization:

  • Increase antibody concentration incrementally

  • Extend primary antibody incubation time or temperature

  • Use enhanced chemiluminescent substrates for greater sensitivity

  • Adjust exposure times for Western blots

• Antigen accessibility problems:

  • For IHC applications, optimize antigen retrieval methods

  • Test different fixation protocols that may better preserve ELP4 epitopes

  • Consider membrane permeabilization adjustments for IHC-F

• Expression level considerations:

  • Verify that your experimental model expresses detectable levels of ELP4

  • Consider using positive control lysates with confirmed ELP4 expression

  • Evaluate if treatment conditions might downregulate ELP4 expression

If standard approaches fail to resolve the issue, consider more advanced techniques such as IP-Western for enrichment of low-abundance ELP4 protein prior to detection with the HRP-conjugated antibody.

What controls are essential when working with ELP4 antibody in various applications?

When designing experiments with ELP4 antibody (HRP conjugated), implementing appropriate controls is crucial for result validation:

• Positive controls:

  • Cell lines with confirmed ELP4 expression: Based on literature and manufacturer validation, HUVEC cells, K562, and ZR-75 cell lines have demonstrated detectable ELP4 expression

  • Recombinant ELP4 protein at known concentrations for standard curves in quantitative applications

  • Tissue sections with established ELP4 expression patterns (e.g., kidney tissues have been used successfully for antibody validation)

• Negative controls:

  • Isotype control: Use an irrelevant IgG of the same isotype as the ELP4 antibody (rabbit IgG for most ELP4 antibodies)

  • Antibody omission control: Process samples without primary antibody to assess secondary reagent specificity

  • Knockout or knockdown samples: When available, use ELP4-knockout cell lines as definitive negative controls, similar to the approach shown for other proteins

• Specificity controls:

  • Peptide competition assay: Pre-incubate antibody with the immunizing peptide to verify signal specificity

  • Cross-species reactivity testing: Validate signals across human, mouse, and rat samples as indicated by the reactivity profile

• Technical controls:

  • Loading controls for Western blot (GAPDH, β-actin)

  • Tissue processing controls for IHC applications

  • Standardized positive samples across experimental batches to control for technical variations

• Subcellular localization controls:

  • Co-staining with subcellular markers (nuclear, cytoplasmic) to confirm the expected dual localization pattern

  • Subcellular fractionation followed by Western blotting to verify compartment-specific signals

Implementation of these comprehensive controls will enhance data reliability and facilitate troubleshooting if unexpected results occur.

How do post-translational modifications of ELP4 affect antibody binding efficiency?

Post-translational modifications (PTMs) of ELP4 can significantly impact antibody recognition and binding efficiency, creating potential challenges for detection:

The ELP4 antibodies referenced are developed against synthetic peptides derived from human ELP4, specifically from regions encompassing amino acids 321-424/424 . PTMs within or near this epitope region can dramatically affect antibody binding through several mechanisms:

• Phosphorylation effects:

  • ELP4 functions within the elongator complex, which is regulated by phosphorylation events

  • Phosphorylation of residues within the antibody epitope can create steric hindrance or alter charge distribution

  • This may prevent antibody recognition, resulting in false-negative results or signal reduction

  • Researchers should consider using phosphatase treatment of samples when phosphorylation is suspected

• Acetylation considerations:

  • Given ELP4's involvement in histone acetyltransferase activity, it may itself be subject to acetylation

  • Acetylation neutralizes positive charges on lysine residues, potentially disrupting antibody-epitope interactions

  • This modification can be particularly relevant when studying ELP4's role in transcriptional regulation

• Ubiquitination and SUMOylation:

  • These larger modifications can completely block antibody access to epitopes

  • They often regulate protein turnover and nuclear-cytoplasmic shuttling, which is relevant given ELP4's dual localization

  • Consider using deubiquitinating enzymes in sample preparation when investigating protein degradation pathways

• Methodological approaches to address PTM interference:

  • Use multiple antibodies targeting different ELP4 epitopes to confirm results

  • Employ immunoprecipitation followed by PTM-specific detection methods

  • Consider mass spectrometry analysis to identify specific modifications

  • Compare native and denaturing conditions to assess structural epitope changes

Understanding these potential modification sites and their impact on antibody recognition is crucial for accurate interpretation of experimental results, particularly in studies examining ELP4 regulation under different cellular conditions.

What cross-reactivity considerations are important when studying ELP4 across species?

When conducting comparative studies of ELP4 across species, researchers must carefully consider cross-reactivity profiles of their antibodies:

• Documented species reactivity:

  • The polyclonal ELP4 antibody (HRP conjugated) has confirmed reactivity with human, mouse, and rat ELP4

  • The recombinant/monoclonal version shows verified reactivity with human and mouse ELP4

  • Predicted reactivity with rabbit ELP4 is indicated for the polyclonal antibody

• Sequence homology analysis:

  • ELP4 shows high conservation across mammalian species

  • Researchers should perform sequence alignments of the antibody epitope region (aa 321-424/424) across target species

  • Higher sequence identity correlates with increased likelihood of cross-reactivity

• Validation strategies for cross-species applications:

  • Perform Western blots on samples from each species to verify band specificity and molecular weight

  • Include species-specific positive controls alongside experimental samples

  • Consider titrating antibody concentrations when working with less validated species

• Potential cross-reactivity with paralogous proteins:

  • ELP4 belongs to the Elongator complex family, which includes related proteins

  • Evaluate sequence similarity between ELP4 and other elongator complex proteins within the epitope region

  • Be cautious of potential signals from related proteins, particularly in less characterized species

• Technical considerations for cross-species work:

  • May require optimization of dilution factors (starting with more concentrated antibody for less validated species)

  • Species-specific secondary antibodies should be considered when not using HRP-conjugated primary antibodies

  • Antigen retrieval protocols may need species-specific adjustments for IHC applications

This comprehensive approach to cross-species validation ensures reliable comparative studies of ELP4 across different model organisms, enhancing translational relevance of research findings.

What are the optimal storage conditions for maintaining ELP4 antibody activity?

Proper storage of ELP4 antibody, HRP conjugated, is critical for maintaining reactivity and specificity over time. The manufacturer recommends the following conditions:

• Temperature requirements:

  • Store at -20°C for long-term preservation

  • Avoid storage at 4°C for extended periods, as this accelerates degradation of both antibody and HRP enzyme

• Buffer composition:

  • The antibody is provided in an aqueous buffered solution containing:

    • 0.01M TBS (pH 7.4)

    • 1% BSA (stabilizer)

    • 0.03% Proclin300 (antimicrobial agent)

    • 50% Glycerol (cryoprotectant)

  • This formulation is designed to maintain stability during freeze-thaw cycles

• Light protection:

  • HRP conjugates are somewhat sensitive to light exposure

  • Store in amber or opaque containers, or wrap tubes in aluminum foil

  • Minimize exposure to direct light during experimental procedures

• Sterility considerations:

  • Use sterile technique when handling to prevent microbial contamination

  • Avoid introducing foreign material that might contain peroxidase activity

• Working stock preparation:

  • For frequent use, consider preparing small working aliquots

  • Maintain sterility and proper buffer conditions in working stocks

  • Document preparation date and storage conditions for each aliquot

Following these storage parameters will maximize antibody shelf life and ensure consistent experimental results throughout the usage period.

How can researchers minimize antibody degradation from freeze-thaw cycles?

Freeze-thaw cycles can significantly impact antibody performance through various degradation mechanisms. The manufacturers specifically advise to "aliquot into multiple vials to avoid repeated freeze-thaw cycles" . Here's a comprehensive strategy to minimize these effects:

• Aliquoting protocol:

  • Upon receipt, divide the stock antibody into single-use aliquots (typically 5-10 μL)

  • Use sterile microcentrifuge tubes for aliquoting

  • Calculate aliquot volumes based on typical experiment needs

  • Label each tube with antibody details, concentration, date, and lot number

• Aliquot storage optimization:

  • Store all aliquots at -20°C as recommended

  • Consider using screw-cap tubes to prevent evaporation during long-term storage

  • Organize storage to minimize the time tubes spend outside freezer during retrieval

• Thawing methodology:

  • Thaw aliquots on ice rather than at room temperature

  • Avoid using heat sources (including hands) to accelerate thawing

  • Once thawed, mix gently by flicking or very brief, gentle vortexing

  • Spin down briefly in a microcentrifuge to collect contents at the bottom

• Working with thawed antibody:

  • Use thawed aliquots immediately for optimal performance

  • Keep on ice while in use

  • Never refreeze a thawed aliquot unless absolutely necessary

• Quality control practices:

  • Include consistent positive controls across experiments to monitor potential degradation

  • Document any observed decrease in signal intensity over time

  • Consider dilution adjustments if loss of activity is observed with older aliquots

The 50% glycerol in the storage buffer provides some cryoprotection, but cannot completely prevent degradation from multiple freeze-thaw cycles. Implementing these practices will maximize antibody performance and consistency throughout your research project.

Which buffer compositions optimize ELP4 antibody performance in different applications?

Buffer compositions significantly influence ELP4 antibody performance across different applications. Here are optimized buffer recommendations:

• Western Blotting:

  • Blocking buffer: 5% non-fat dry milk or 3-5% BSA in TBST (TBS + 0.1% Tween-20)

  • Antibody dilution buffer: 1% BSA in TBST, which maintains consistency with the antibody storage buffer

  • Wash buffer: TBST (TBS + 0.1% Tween-20)

  • Stripping buffer (if needed): Mild stripping with glycine (pH 2.2) rather than harsh β-mercaptoethanol-based buffers

• Immunohistochemistry:

  • Antigen retrieval buffer: Citrate buffer (10mM, pH 6.0) for HIER

  • Blocking buffer: 1-3% BSA in PBS with 0.1% Triton X-100

  • Antibody dilution buffer: 1% BSA in PBS with 0.01% Triton X-100

  • Wash buffer: PBS with 0.05% Tween-20

• ELISA:

  • Coating buffer: Carbonate-bicarbonate buffer (pH 9.6)

  • Blocking buffer: 2% BSA in PBS

  • Antibody dilution buffer: 0.5-1% BSA in PBS with 0.05% Tween-20

  • Wash buffer: PBS with 0.05% Tween-20

• Buffer additives to consider:

  • Protease inhibitors: Include in lysis and sample preparation buffers

  • Phosphatase inhibitors: Important when studying potential ELP4 phosphorylation

  • EDTA (1mM): Can help reduce background in some applications

  • Sodium azide (0.02%): For preserving non-HRP conjugated antibodies (NOT recommended with HRP conjugates)

• pH considerations:

  • Maintain buffer pH between 7.2-7.6 for most applications

  • The antibody is formulated at pH 7.4 , suggesting optimal activity near physiological pH

These buffer compositions balance the needs for antibody stability, specificity, and accessibility to target epitopes in different experimental contexts while minimizing background signals.

What is the expected stability timeline for ELP4 antibody, HRP conjugated?

Understanding the stability timeline of ELP4 antibody with HRP conjugation is essential for experimental planning and resource management:

• Shelf-life parameters:

  • Manufacturer storage: Typically 12-18 months when stored as recommended

  • Researcher storage: Approximately 12 months at -20°C in manufacturer's buffer when properly aliquoted

  • Working solution: 1-2 weeks at 4°C once diluted in working buffer

• Stability indicators by application:

  • Western blotting: Earliest signs of degradation typically manifest as reduced signal intensity and increased background

  • IHC applications: May show diminished staining intensity and specificity

  • ELISA: Decreased linear range and higher variability between technical replicates

• HRP conjugate-specific considerations:

  • HRP enzyme activity typically degrades faster than antibody binding capacity

  • Loss of signal may occur while antigen recognition remains intact

  • Signal reduction of 10-20% per year can be expected even with optimal storage

• Stability monitoring approach:

  • Regular testing with consistent positive control samples

  • Documentation of signal intensity over time

  • Adjustment of dilution factors to compensate for gradual activity loss

  • Comparison of new and older lots using standardized samples

• Extending functional lifespan:

  • Strict adherence to storage at -20°C

  • Maintenance of sterile conditions

  • Addition of proper stabilizing proteins (BSA already included in manufacturer's formulation)

  • Protection from light exposure

With proper handling and storage practices, researchers can expect to maintain reliable performance from ELP4 antibody, HRP conjugated, for approximately 12 months from receipt date, with gradual diminishment of signal intensity rather than sudden loss of functionality.

Can ELP4 antibody, HRP conjugated be effectively used in multiplex assays?

ELP4 antibody, HRP conjugated, presents both opportunities and limitations for multiplex assay applications:

• Compatibility with multiplexing techniques:

  • Chromogenic multiplexing: Limited compatibility due to HRP's standard brown/red DAB precipitation reaction

  • Fluorescent multiplex IHC: Not directly compatible without additional steps, as HRP conjugates are designed for chromogenic detection

  • Sequential multiplexing: Can be incorporated into sequential staining protocols with proper inactivation steps

• Methodological approaches for multiplexing with HRP-conjugated antibodies:

  • Tyramide signal amplification (TSA) conversion: The HRP can be used to deposit fluorescent tyramide, converting the signal to fluorescence

  • Sequential staining with heat-mediated antibody stripping between rounds

  • Spectral unmixing when using different chromogens for multiple HRP-conjugated antibodies

• Recommended co-detection targets with ELP4:

  • RNA Polymerase II markers to study co-localization with elongation complex

  • Nuclear markers to confirm subcellular localization

  • Other elongator complex components to study complex assembly

• Technical considerations for multiplexing:

  • Order of antibody application: Apply HRP-conjugated ELP4 antibody first in sequential protocols

  • Complete HRP inactivation: Use 3% hydrogen peroxide treatment between rounds

  • Signal separation: Ensure spectral or spatial separation of multiple signals

• Quantitative multiplexing platforms:

  • Compatible with quantitative chromogenic multiplexed IHC systems after protocol optimization

  • May require custom secondary detection when used with platforms like Vectra/Polaris systems

While the direct HRP conjugation presents certain limitations for traditional fluorescent multiplexing, creative methodological approaches can incorporate ELP4 antibody, HRP conjugated, into various multiplex experimental designs with proper optimization and controls.

What methods should be used to validate ELP4 antibody specificity?

Comprehensive validation of ELP4 antibody specificity is essential for generating reliable research data. The following methodological approaches are recommended:

• Genetic validation methods:

  • CRISPR/Cas9 knockout: Generate ELP4 knockout cell lines for definitive negative controls

  • siRNA/shRNA knockdown: Create cells with reduced ELP4 expression to demonstrate signal correlation with expression level

  • Overexpression systems: Transfect cells with ELP4 expression constructs to show signal enhancement

• Biochemical validation approaches:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

  • Immunoprecipitation followed by mass spectrometry: Confirm pulled-down protein identity

  • Orthogonal detection: Compare results with alternative antibodies targeting different ELP4 epitopes

• Technical validation strategies:

  • Western blot analysis: Verify single band of expected molecular weight (~50-55 kDa)

  • Cross-species reactivity testing: Confirm signal in human, mouse, and rat samples as indicated

  • Immunohistochemistry pattern analysis: Compare with established ELP4 expression patterns

• Multi-application concordance:

  • Compare results across different techniques (WB, IHC, ELISA)

  • Assess correlation between protein detection and mRNA expression data

  • Evaluate detection in tissues with known high and low ELP4 expression

• Literature-based validation:

  • Compare detected expression patterns with published RNA-seq data

  • Assess subcellular localization against reported cytoplasmic and nuclear distribution

  • Evaluate interaction with known binding partners

Which experimental models best represent physiological ELP4 expression and function?

Selecting appropriate experimental models is crucial for studying physiological ELP4 expression and function:

• Cell line models for ELP4 research:

  • HUVEC cells: Demonstrated reliable ELP4 expression in validation studies

  • K562 cells: Human chronic myelogenous leukemia cell line with verified ELP4 expression

  • ZR-75 cells: Human breast cancer cell line with detectable ELP4 levels

  • COLO 205: Human colorectal adenocarcinoma cells with confirmed expression

  • Neural cell lines: Particularly relevant given ELP4's association with neurodevelopmental conditions

• Tissue models for studying ELP4:

  • Kidney tissue: Successfully used for antibody validation in IHC applications

  • Brain tissue: Relevant for studying ELP4's role in neurological development

  • Tissues with active transcription: Important for examining ELP4's role in the elongator complex

• Animal models for in vivo studies:

  • Mouse models: Confirmed cross-reactivity with mouse ELP4

  • Rat models: Verified for polyclonal antibody applications

  • Consider developmental timepoints, as ELP4 functions may be particularly critical during development

• Disease-relevant models:

  • Epilepsy models: ELP4 variations have been associated with epilepsy risk

  • Neurodevelopmental disorder models: Relevant for studying ELP4's role in brain development

  • Cancer models: For investigating potential roles in transcriptional dysregulation

• Experimental manipulation strategies:

  • Elongator complex perturbation: Study ELP4 in context of complex assembly/disassembly

  • Stress response induction: Examine ELP4 dynamics under cellular stress conditions

  • Developmental trajectory analysis: Track ELP4 expression across developmental stages

When selecting models, researchers should consider both the specific aspect of ELP4 biology under investigation and the validated reactivity profile of their antibody. Comparative analysis across multiple models can provide the most comprehensive understanding of physiological ELP4 function.

How does ELP4 detection correlate with RNA polymerase II elongation complex activity?

Understanding the relationship between ELP4 detection and RNA polymerase II elongation complex activity provides important context for interpreting experimental results:

• Functional correlation:

  • ELP4 "acts as subunit of the RNA polymerase II elongator complex, which is a histone acetyltransferase component of the RNA polymerase II (Pol II) holoenzyme and is involved in transcriptional elongation"

  • Detection levels may reflect active transcription states in cells or tissues

  • Co-detection with phosphorylated RNA Pol II (Ser2-P) can indicate active elongation complexes

• Mechanistic relationships:

  • Chromatin remodeling function: ELP4 "may play a role in chromatin remodeling and is involved in acetylation of histones H3 and probably H4" , facilitating Pol II progression

  • tRNA modification role: ELP4 "is required for multiple tRNA modifications, including mcm5U, mcm5s2U, and ncm5U" , which may indirectly affect translation of Pol II products

  • These dual functions suggest analyzing both nuclear and cytoplasmic ELP4 pools

• Experimental approaches for correlation studies:

  • Chromatin immunoprecipitation (ChIP): Assess ELP4 localization at actively transcribed genes

  • Co-immunoprecipitation: Determine physical association with Pol II complex components

  • Transcription inhibition experiments: Examine ELP4 dynamics following treatment with transcriptional inhibitors

• Quantitative relationships:

  • Stoichiometric considerations: ELP4 detection may not directly correlate with Pol II levels

  • Dynamic regulation: Post-translational modifications may affect detection without changing absolute protein levels

  • Spatial organization: Nuclear/cytoplasmic distribution shifts may reflect functional state changes

• Research applications:

  • Developmental studies: Examine correlation during high-transcription developmental phases

  • Disease models: Investigate potential dysregulation in conditions with transcriptional defects

  • Therapeutic intervention assessment: Monitor ELP4 as a potential biomarker for treatments targeting transcriptional machinery

This complex relationship between ELP4 detection and elongation complex activity provides a conceptual framework for designing experiments that move beyond mere detection to functional characterization of this important component of transcriptional regulation.