ELP4 Antibody, FITC conjugated

What is ELP4 and why is it significant in research?

ELP4 (Elongator Complex Protein 4) functions as a subunit of the RNA polymerase II elongator complex, which is a histone acetyltransferase component of the RNA polymerase II (Pol II) holoenzyme involved in transcriptional elongation. This protein plays crucial roles in chromatin remodeling and participates in the acetylation of histones H3 and likely H4 . Additionally, ELP4 is an essential component of the elongator complex required for multiple tRNA modifications, including mcm5U (5-methoxycarbonylmethyl uridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), and ncm5U . Its location in both the cytoplasm and nucleus suggests multiple functional roles in cellular processes, making it an important target for epigenetic and transcriptional regulation studies.

What is the difference between polyclonal and monoclonal ELP4 antibodies with FITC conjugation?

The primary distinction lies in antibody production and epitope recognition. Polyclonal ELP4 antibodies with FITC conjugation (such as catalog #bs-14574R-FITC) are derived from multiple B-cell lineages and recognize various epitopes on the ELP4 protein, offering broader detection capabilities . In contrast, monoclonal ELP4 antibodies with FITC conjugation (like catalog #bsm-62511r-fitc) are produced from a single B-cell clone and recognize a specific epitope, providing higher specificity but potentially lower sensitivity . For research applications requiring comprehensive detection of ELP4 variants, polyclonal antibodies may be preferable, while applications demanding precise detection of specific ELP4 epitopes would benefit from monoclonal antibodies.

What applications are appropriate for ELP4 antibody with FITC conjugation?

ELP4 antibodies with FITC conjugation can be utilized across multiple experimental applications. The polyclonal variant (bs-14574R-FITC) has been validated for Western Blotting (WB) at dilutions of 1:300-5000, as well as for various immunofluorescence applications including IHC-P, IHC-F, and ICC at dilutions of 1:50-200 . The monoclonal variant (bsm-62511r-fitc) is suitable for Western Blotting and ICC immunofluorescence . Additionally, FITC-conjugated antibodies are broadly applicable for flow cytometry, allowing researchers to identify and enumerate specific protein-expressing cells within heterogeneous populations . The spectral characteristics of FITC make it compatible with standard FITC filter sets (excitation ~495 nm, emission ~520 nm) on most fluorescence microscopes and flow cytometers.

What are the optimal storage conditions for FITC-conjugated ELP4 antibodies?

For maximum stability and retention of fluorescence intensity, FITC-conjugated ELP4 antibodies should be stored at -20°C in their provided storage buffers . These buffers typically contain cryoprotectants such as glycerol (50%) to prevent freeze-thaw damage, BSA (1%) for protein stability, and antimicrobial agents like Proclin300 (0.02-0.03%) to prevent microbial growth . It is crucial to aliquot the antibody into multiple vials upon receipt to avoid repeated freeze-thaw cycles, which can degrade both the antibody and the FITC fluorophore. When working with the antibody, keep it protected from prolonged light exposure, as FITC is susceptible to photobleaching. For short-term storage during experimental procedures, keep the antibody on ice and in the dark.

What species reactivity should be expected from ELP4 antibodies?

The reactivity profile varies between different ELP4 antibody products. The polyclonal antibody (bs-14574R-FITC) demonstrates confirmed reactivity with human, mouse, and rat samples, with predicted reactivity to rabbit samples based on sequence homology . The monoclonal variant (bsm-62511r-fitc) has confirmed reactivity with human and mouse samples . When working with species not listed in the confirmed reactivity panel, researchers should conduct preliminary validation experiments or consult the manufacturer regarding sequence homology predictions. Cross-reactivity validation is particularly important when studying ELP4 in evolutionarily distant model organisms.

How can I optimize immunofluorescence protocols using FITC-conjugated ELP4 antibodies?

Optimizing immunofluorescence with FITC-conjugated ELP4 antibodies requires attention to several key parameters. For cell preparations, fix cells with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100 or saponin to facilitate nuclear access, as ELP4 localizes to both cytoplasm and nucleus . Begin with the manufacturer's recommended dilution ranges (typically 1:50-200 for immunofluorescence applications) , then perform a titration experiment to determine optimal signal-to-noise ratio for your specific cell type or tissue. For tissue sections, antigen retrieval methods (citrate buffer, pH 6.0) may enhance epitope accessibility. Include appropriate blocking steps (5-10% normal serum from the same species as secondary antibody or 1-3% BSA) to minimize non-specific binding. To counteract FITC's susceptibility to photobleaching, incorporate anti-fade mounting media containing DAPI for nuclear counterstaining. For co-localization studies, select secondary fluorophores with minimal spectral overlap with FITC's emission spectrum.

What considerations are important when using ELP4-FITC antibodies for flow cytometry?

When employing FITC-conjugated ELP4 antibodies for flow cytometry, several technical factors require attention. For optimal staining, titrate the antibody to determine the ideal concentration, typically ≤0.5 μg antibody per million cells . Since ELP4 localizes to both cytoplasm and nucleus, effective permeabilization is essential—use commercial permeabilization buffers compatible with intracellular targets or saponin-based solutions (0.1-0.5%). Include appropriate blocking steps to reduce non-specific binding and background fluorescence. Always run proper controls including:

• Unstained cells for autofluorescence assessment

• Isotype-matched FITC-conjugated controls at equal concentrations to evaluate non-specific binding

• Blocking controls where cells are pre-incubated with unconjugated antibody before staining with the FITC-conjugated version

Compensation is critical when using multiple fluorophores, as FITC's emission spectrum may overlap with other common fluorophores like PE. Consider using viability dyes compatible with fixed cells to exclude dead cells, which can bind antibodies non-specifically.

How do I determine the specificity of my ELP4-FITC antibody results?

Validating the specificity of ELP4-FITC antibody results requires multiple complementary approaches. First, compare the observed subcellular localization with established patterns for ELP4 (cytoplasmic and nuclear distribution) . Second, implement appropriate controls including isotype controls, secondary-only controls (for indirect methods), and competitive blocking using unlabeled antibodies of the same clone . For definitive validation, consider using:

• Positive controls: Cell lines with known ELP4 expression (based on literature or database information)

• Negative controls: ELP4 knockdown or knockout cell systems using siRNA or CRISPR/Cas9

• Orthogonal methods: Corroborate immunofluorescence findings with Western blot or mass spectrometry data

In addition, cross-validation using a second anti-ELP4 antibody targeting a different epitope can provide further confidence in the specificity of your observations. For complex samples, pre-adsorption tests where the antibody is pre-incubated with purified ELP4 protein before staining can help determine binding specificity.

What is the recommended protocol for using ELP4-FITC antibodies in multicolor immunofluorescence?

For multicolor immunofluorescence utilizing ELP4-FITC antibodies alongside other markers, a sequential staining approach is recommended:

• Sample Preparation:

  • For cultured cells: Fix with 4% paraformaldehyde (15 minutes), permeabilize with 0.2% Triton X-100 (10 minutes)

  • For tissue sections: Deparaffinize, rehydrate, and perform antigen retrieval (citrate buffer pH 6.0)

• Blocking:

  • Incubate samples in 5% normal serum (from secondary antibody host species) with 1% BSA for 60 minutes

• Primary Antibody Incubation:

  • Apply ELP4-FITC antibody at optimal dilution (1:50-200)

  • For co-staining, use primary antibodies raised in different host species

  • Incubate overnight at 4°C in a humidified chamber

• Washing:

  • Three 5-minute washes with PBS/0.1% Tween-20

• Nuclear Counterstaining:

  • DAPI (1 μg/ml) for 5 minutes

• Mounting:

  • Use anti-fade mounting medium to preserve FITC fluorescence

When selecting companion fluorophores, choose those with minimal spectral overlap with FITC (e.g., Cy5, Alexa 647) for clearer signal separation. Document control samples stained with individual antibodies to establish baseline signal intensity and distribution for comparison with multiplex images.

How can I enhance FITC signal in low-abundance ELP4 detection scenarios?

For detecting low-abundance ELP4 expression, several signal amplification strategies can be employed:

• Tyramide Signal Amplification (TSA):

  • Use biotinylated anti-FITC antibodies followed by streptavidin-HRP

  • Apply tyramide-FITC substrate for signal amplification (10-100 fold increase)

• Anti-FITC Antibody Enhancement:

  • Apply anti-FITC antibodies conjugated to additional FITC molecules or brighter fluorophores

  • This provides 2-5 fold signal enhancement while maintaining specificity

• Extended Exposure Times:

  • For imaging, use longer exposure times with cameras with high sensitivity and low noise characteristics

  • For flow cytometry, adjust PMT voltage to optimize FITC detection

• Specialized Mounting Media:

  • Use enhanced anti-fade reagents specifically formulated for FITC preservation

  • Some commercial products contain signal enhancers that increase apparent brightness

• Sample Preparation Optimization:

  • Extend primary antibody incubation time to 48-72 hours at 4°C

  • Use gentle detergents (0.01% saponin) for improved antibody penetration

The optimal approach depends on your specific application and sample type. For quantitative studies, validate that signal amplification methods maintain the linear relationship between signal intensity and target abundance.

How can I address high background issues when using ELP4-FITC antibodies?

High background is a common challenge when using FITC-conjugated antibodies. The following systematic approach can help resolve this issue:

After implementing these adjustments, always include a matched isotype control antibody with FITC conjugation at the same concentration to establish baseline background levels. Additionally, consider acquisition settings: for microscopy, adjust exposure to minimize background while preserving specific signal; for flow cytometry, set compensation correctly to account for FITC spillover into other channels .

What are the best approaches for quantifying ELP4 expression using FITC-conjugated antibodies?

Accurate quantification of ELP4 expression using FITC-conjugated antibodies requires appropriate methods depending on the experimental platform:

For Flow Cytometry Quantification:

• Use calibration beads with defined FITC fluorescence to establish a standard curve

• Report data as Molecules of Equivalent Soluble Fluorochrome (MESF) rather than arbitrary units

• Include a quantitative flow cytometry standard for each experiment

• Calculate Mean/Median Fluorescence Intensity (MFI) after subtracting isotype control values

For Microscopy-Based Quantification:

• Capture images with identical acquisition settings across all samples

• Use software like ImageJ/FIJI, CellProfiler, or QuPath for automated analysis

• Define regions of interest (ROI) for nuclear and cytoplasmic compartments separately

• Measure integrated density rather than mean intensity to account for total protein content

• Include internal reference standards in each sample for normalization

For Western Blot Quantification (after stripping and reprobing FITC-labeled membranes):

• Use fluorescence-compatible membrane and scanner

• Include standard curve of recombinant ELP4 protein on each blot

• Normalize to appropriate loading controls (β-actin, GAPDH, or total protein stain)

In all quantification approaches, biological and technical replicates are essential for statistical validity, and appropriate normalization controls must be included to account for experimental variability.

How do I interpret contradictory results between different applications using ELP4-FITC antibodies?

When facing contradictory results across different applications (e.g., IF showing nuclear localization while WB indicates different molecular weight), consider the following systematic analysis approach:

• Examine epitope accessibility:

  • Different applications expose different epitopes

  • Native protein folding, fixation methods, and denaturing conditions affect epitope recognition

  • The ELP4 epitope range (321-424/424) may be differentially accessible in various applications

• Consider post-translational modifications:

  • ELP4 may undergo modifications affecting antibody recognition

  • Different cell types or experimental conditions may alter ELP4's modification state

  • Verify if discrepancies correlate with treatment conditions affecting post-translational modifications

• Evaluate isoform detection:

  • Check if the antibody recognizes all ELP4 isoforms or specific variants

  • Compare observed molecular weights with predicted values for known isoforms

  • Consider tissue-specific or condition-dependent expression of different isoforms

• Validate with orthogonal methods:

  • Use non-antibody-based methods (mass spectrometry, RNA-seq)

  • Apply alternative antibodies targeting different ELP4 epitopes

  • Implement genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity

Resolution often requires triangulation of multiple methods and carefully controlled experiments to determine which results most accurately reflect the biological reality of ELP4 expression and localization in your specific experimental system.

How can ELP4-FITC antibodies be utilized in studies of transcriptional elongation mechanisms?

ELP4-FITC antibodies provide valuable tools for investigating transcriptional elongation mechanisms through several sophisticated approaches:

• Chromatin Immunoprecipitation followed by Fluorescence Microscopy (ChIP-FM):

  • Use ELP4-FITC antibodies to visualize chromatin association patterns

  • Combine with antibodies against RNA Polymerase II to assess co-localization

  • Quantify spatial relationships between ELP4 and active transcription sites

• Dynamic Tracking in Live Cells:

  • For cell lines with low phototoxicity sensitivity, use mild fixation protocols that preserve FITC fluorescence

  • Employ spinning disk confocal microscopy to capture ELP4 dynamics during transcriptional activation

  • Correlate with markers of transcriptional activity using multi-channel acquisition

• Super-Resolution Microscopy:

  • Apply techniques like Structured Illumination Microscopy (SIM) or Stochastic Optical Reconstruction Microscopy (STORM)

  • Resolve nanoscale organization of ELP4 relative to transcription factories

  • Quantify clustering patterns under different transcriptional states

The Elongator complex's role in histone acetylation makes these approaches particularly valuable for understanding how ELP4 contributes to chromatin remodeling during transcriptional elongation, potentially revealing novel regulatory mechanisms in gene expression control.

What are the considerations for using ELP4-FITC antibodies in studies of tRNA modification pathways?

When investigating ELP4's role in tRNA modification pathways using FITC-conjugated antibodies, researchers should address several specialized considerations:

• Co-localization with tRNA Processing Machinery:

  • Pair ELP4-FITC antibodies with markers of tRNA processing bodies

  • Assess spatial relationships with enzymes involved in mcm5U, mcm5s2U, and ncm5U modifications

  • Quantify colocalization coefficients (Pearson's, Mander's) under various cellular conditions

• Stress Response Analysis:

  • Monitor ELP4 localization changes during translation stress

  • Compare localization patterns before and after exposure to translation inhibitors

  • Correlate with markers of stress granules and P-bodies

• Cell Cycle Dependency:

  • Synchronize cells and examine ELP4 distribution across cell cycle phases

  • Determine if tRNA modification activity correlates with specific cell cycle stages

  • Use flow cytometry to correlate ELP4-FITC signal intensity with cell cycle markers

• Kinetic Studies:

  • Implement pulse-chase experiments to track newly synthesized ELP4

  • Correlate with tRNA modification activity using specialized radiometric assays

  • Determine temporal relationships between ELP4 expression, localization, and tRNA modification levels

These approaches can provide insights into how the Elongator complex coordinates tRNA modifications, which are critical for translational accuracy and efficiency in both normal cellular function and disease states.

What emerging technologies might enhance the utility of ELP4-FITC antibodies in research?

Several cutting-edge technologies are poised to expand the applications of ELP4-FITC antibodies in advanced research settings:

• Spatial Transcriptomics Integration:

  • Combining ELP4-FITC immunofluorescence with spatial transcriptomics

  • Correlating ELP4 protein localization with gene expression patterns in the same tissue section

  • Revealing relationships between ELP4 distribution and transcriptional landscapes

• Mass Cytometry Adaptation:

  • Developing metal-conjugated ELP4 antibodies compatible with CyTOF technology

  • Enabling high-dimensional analysis of ELP4 in relation to dozens of other proteins simultaneously

  • Providing deeper insights into ELP4's role in complex cellular hierarchies

• Proximity Labeling Applications:

  • Engineering ELP4 fusion proteins with proximity-dependent biotin ligases

  • Identifying novel interaction partners of ELP4 in living cells

  • Defining the dynamic interactome of ELP4 under various cellular conditions

• Automated High-Content Screening:

  • Implementing ELP4-FITC antibodies in high-throughput imaging platforms

  • Screening compound libraries for modulators of ELP4 localization or expression

  • Discovering novel regulators of Elongator complex function

These technological advances, when applied with ELP4-FITC antibodies, will likely reveal new dimensions of ELP4 biology and its roles in transcriptional regulation, tRNA modification, and cellular homeostasis.

How might ELP4-FITC antibodies contribute to understanding disease mechanisms?

ELP4-FITC antibodies offer promising applications for elucidating disease mechanisms across several pathological contexts:

• Neurodevelopmental Disorders:

  • ELP4's proximity to PAX6 (as indicated by its synonym PAX6 Neighbor Gene Protein) suggests potential involvement in neurodevelopmental processes

  • ELP4-FITC antibodies can help characterize expression patterns in neuronal subtypes during development

  • Comparative analyses between normal and pathological brain tissues may reveal altered ELP4 distribution

• Cancer Biology:

  • Dysregulation of transcriptional elongation is implicated in multiple cancer types

  • ELP4-FITC antibodies enable assessment of altered subcellular localization in tumor samples

  • Flow cytometric analysis with ELP4-FITC can identify potential cancer cell subpopulations with distinctive ELP4 expression profiles

• Translational Fidelity Disorders:

  • ELP4's role in tRNA modification links it to diseases involving translational fidelity

  • Comparative analysis of ELP4 localization in cells from patients with translation-related disorders

  • Correlation of ELP4 distribution patterns with specific disease phenotypes