ALYREF Antibody, Biotin conjugated

What is ALYREF and why is it important in molecular biology research?

ALYREF (Aly/REF export factor) is a 26.9 kDa protein that serves as a critical component of the THO complex, playing essential roles in mRNA processing and nuclear export. This protein, also referred to as Aly, tho4, BEF, REF, or THO complex subunit 4, functions as an adaptor molecule that connects the mRNA export machinery with transcription and RNA processing events . The importance of ALYREF in research stems from its fundamental role in linking RNA processing with nuclear export mechanisms. ALYREF has been shown to interact with m5C-modified mRNAs, stabilizing specific transcripts such as TBL1XR1 and KMT2E, which has significant implications for gene expression regulation . For researchers investigating RNA metabolism, splicing mechanisms, or mRNA trafficking, ALYREF represents a key target for understanding these complex cellular processes.

What experimental applications are supported by biotin-conjugated ALYREF antibodies?

Biotin-conjugated ALYREF antibodies support multiple experimental applications with specific technical advantages. The primary applications include:

Unlike unconjugated antibodies, biotin-conjugated ALYREF antibodies offer enhanced flexibility in detection systems, particularly advantageous for detecting low-abundance proteins or when performing multiplexed experiments . The biotin-streptavidin interaction provides one of the strongest non-covalent biological interactions, allowing for amplification of detection signals and improved sensitivity in challenging experimental systems. Researchers should note that optimal dilutions are assay-dependent and antibody lot-specific, necessitating optimization for each experimental setup.

How is the specificity of ALYREF antibodies validated for research applications?

Validating the specificity of ALYREF antibodies requires multiple complementary approaches to ensure experimental reliability. Current best practices include:

• Target knockout/knockdown verification: Comparing antibody reactivity in wild-type cells versus ALYREF-depleted cells (through CRISPR/Cas9 knockout or siRNA knockdown) to confirm signal specificity .

• Cross-reactivity testing: Evaluating reactivity across multiple species (human, mouse, rat) to confirm conservation-based recognition patterns. Published data confirms cross-reactivity with human, mouse, rat, and pig ALYREF proteins .

• Molecular weight verification: Confirming detection at the expected molecular weight (calculated at 27 kDa, though typically observed at 27-30 kDa due to post-translational modifications) .

• Multi-application concordance: Validating consistent results across different applications (WB, IF, IHC) to ensure epitope recognition in various protein conformations and sample preparation techniques.

• Blocking peptide competition: Conducting competitive binding assays with the immunizing peptide to demonstrate binding specificity to the target epitope.

Researchers should note that even validated antibodies can exhibit batch-to-batch variation, making it essential to maintain consistent validation protocols throughout a research project. For biotin-conjugated ALYREF antibodies specifically, additional controls for endogenous biotin should be included to prevent false-positive results, particularly in tissues with high biotin content.

What are the optimal fixation and antigen retrieval methods for ALYREF detection in immunohistochemistry?

Successful ALYREF detection in immunohistochemistry requires careful consideration of fixation and antigen retrieval methods to preserve epitope accessibility. Based on published protocols, the following approaches yield optimal results:

The efficacy of high pH (pH 9.0) antigen retrieval for ALYREF detection is particularly noteworthy, as it typically yields superior results compared to citrate buffer methods. This difference likely reflects the need to effectively expose the epitope recognized by the antibody while preserving the nuclear localization context of ALYREF . Researchers should be aware that ALYREF predominantly displays nuclear localization with some nucleolar enrichment, presenting a characteristic staining pattern that serves as an internal quality control for antibody performance.

How should researchers design RNA immunoprecipitation (RIP) experiments using biotin-conjugated ALYREF antibodies?

RNA immunoprecipitation (RIP) with biotin-conjugated ALYREF antibodies requires careful experimental design to capture authentic protein-RNA interactions while minimizing experimental artifacts. The recommended protocol follows these key steps:

• Cross-linking optimization: ALYREF-RNA interactions typically require formaldehyde cross-linking (1% for 10 minutes at room temperature). Over-crosslinking can reduce antibody accessibility to epitopes, while under-crosslinking may fail to capture transient interactions.

• Cell lysis conditions: Use gentle non-ionic detergent buffers (e.g., 0.5% NP-40 in PBS with protease inhibitors and RNase inhibitors) to preserve protein-RNA complexes while effectively lysing cells.

• Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding. This step is crucial for reducing background in subsequent analyses.

• Antibody immobilization: For biotin-conjugated antibodies, pre-immobilize on streptavidin-coated magnetic beads (typically 2-5 μg antibody per experiment) before adding to pre-cleared lysates.

• Immunoprecipitation conditions: Incubate at 4°C for 4-6 hours with gentle rotation. Extended incubation can increase yield but may also increase non-specific interactions.

• Washing stringency: Use progressively more stringent washing buffers to remove non-specific interactions while preserving authentic ALYREF-RNA complexes. Typically, 3-5 washes are performed.

• RNA extraction and analysis: Reverse cross-links (65°C for 45 minutes in reverse cross-linking buffer) before RNA extraction, followed by qRT-PCR, RNA-seq, or other analytical methods.

Research has demonstrated that ALYREF specifically recognizes m5C-modified mRNAs, particularly binding to transcripts like TBL1XR1 and KMT2E to regulate their stability . When designing RIP experiments targeting these or other potential ALYREF-associated transcripts, researchers should include appropriate controls, such as IgG control antibodies and input samples, to accurately quantify enrichment. Additionally, validation through complementary methods, such as RNA pulldown assays followed by Western blotting for ALYREF, strengthens the reliability of identified interactions.

What controls are essential when using biotin-conjugated ALYREF antibodies in Western blot applications?

Rigorous controls are essential when using biotin-conjugated ALYREF antibodies in Western blot applications to ensure result validity and interpretability. The following controls should be incorporated:

• Positive control: Include lysates from cells known to express ALYREF at detectable levels (e.g., HeLa, HEK-293, or A431 cells) to confirm antibody functionality .

• Negative control: Utilize lysates from ALYREF-depleted cells (through siRNA knockdown or CRISPR/Cas9 knockout) to verify signal specificity.

• Loading control: Probing for housekeeping proteins (e.g., GAPDH, β-actin) ensures equal loading across samples and permits accurate quantification of relative ALYREF levels.

• Biotin blocking control: Include a lane treated with avidin/streptavidin blocking reagent prior to antibody incubation to identify potential signals from endogenous biotinylated proteins.

• Molecular weight marker: Always include a molecular weight marker to confirm detection at the expected size (27-30 kDa for ALYREF) .

• Secondary-only control: Omit primary antibody but include streptavidin-conjugated detection reagent to identify non-specific binding of the detection system.

• Non-specific band identification: Non-ALYREF-specific bands can be identified by comparing pattern changes between control and ALYREF-depleted samples.

How can researchers optimize co-immunoprecipitation protocols to study ALYREF's protein interaction network?

Optimizing co-immunoprecipitation (co-IP) protocols for studying ALYREF's protein interactome requires careful consideration of buffer conditions and experimental parameters to preserve authentic interactions while minimizing artifacts. The following methodological approach is recommended:

• Buffer optimization: Use low-stringency lysis buffers (e.g., 0.3% CHAPS or 0.5% NP-40 in TBS with protease inhibitors) to preserve weak or transient interactions. High-salt or high-detergent conditions may disrupt relevant protein-protein interactions.

• Cross-linking considerations: For transient interactions, mild cross-linking with DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM for 30 minutes preserves complexes while remaining reversible under reducing conditions.

• Nuclear extraction protocol: Since ALYREF is predominantly nuclear, efficient nuclear extraction is crucial. Use a gentle nuclear extraction buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol) with DNase I treatment to release chromatin-associated complexes.

• Bead selection: For biotin-conjugated antibodies, streptavidin-coated magnetic beads provide superior performance compared to agarose beads, with lower background and better recovery.

• RNA-dependent interactions: To distinguish between RNA-dependent and direct protein-protein interactions, perform parallel experiments with and without RNase A/T1 treatment.

• Validation approach: Confirm interactions through reciprocal co-IP experiments (IP with antibodies against interacting partners and blot for ALYREF) and proximity ligation assays.

ALYREF has been shown to interact with multiple protein partners, including components of the TREX complex (UAP56/DDX39B, CIP29) and mRNA export factors (NXF1/TAP). Recent research has also revealed interactions with RNA modification readers and writers, particularly in the context of cancer progression and chemotherapy resistance . When investigating novel ALYREF interactions, researchers should consider both structural domains of ALYREF that might mediate these interactions: the N-terminal RRM domain (RNA binding) and the C-terminal UBM domain (protein-protein interactions).

What are the key considerations for multiplexed immunofluorescence using biotin-conjugated ALYREF antibodies?

Multiplexed immunofluorescence with biotin-conjugated ALYREF antibodies requires strategic planning to avoid detection system interference while maximizing signal specificity. The following methodological approach addresses these challenges:

• Antibody panel design: When combining biotin-conjugated ALYREF antibodies with other antibodies, carefully plan the detection system to avoid cross-reactivity. Consider using fluorophore-conjugated streptavidin that spectrally separates from other secondary antibodies in the panel.

• Sequential staining protocol: For complex multiplexing, implement a sequential staining approach:

  • First round: ALYREF detection with biotin-conjugated primary antibody

  • Complete with fluorophore-conjugated streptavidin

  • Block remaining biotin binding sites with excess biotin/avidin

  • Proceed with subsequent antibody staining rounds

• Spectral considerations: Select fluorophores with minimal spectral overlap and implement appropriate compensation controls when using detection systems with similar emission spectra.

• Tyramide signal amplification integration: For low-abundance targets co-stained with ALYREF, consider tyramide signal amplification with biotin-conjugated ALYREF antibody, which provides 10-50x signal enhancement through covalent fluorophore deposition.

• Autofluorescence management: Include unstained controls to establish baseline autofluorescence, particularly important in tissues with high intrinsic fluorescence (e.g., liver, brain). Implement autofluorescence quenching methods (e.g., Sudan Black B treatment) as needed.

• Bleed-through controls: Include single-color controls to establish proper exposure settings and confirm absence of channel bleed-through before analyzing co-localization.

• Co-localization analysis: Implement quantitative co-localization metrics (Pearson's correlation coefficient, Manders' overlap coefficient) rather than relying on visual assessment alone.

When studying ALYREF's co-localization with other proteins, it's important to note that ALYREF exhibits dynamic localization patterns, predominantly nuclear with enrichment in nuclear speckles and nucleoli . This localization pattern can change in response to cellular stress, particularly those affecting RNA metabolism. For instance, in cancer cells, altered ALYREF localization may correlate with changes in its interactions with m5C-modified RNAs and other regulatory proteins . These dynamic properties should be considered when interpreting co-localization data.

How does ALYREF's role in RNA metabolism impact experimental design for cancer research applications?

ALYREF's emerging roles in RNA metabolism and cancer progression necessitate specialized experimental approaches when investigating its contributions to oncogenic mechanisms. The following methodological considerations are crucial:

• Cell line model selection: When studying ALYREF in cancer contexts, researchers should carefully select appropriate cell line models. Recent studies have demonstrated that ALYREF expression is significantly elevated in oxaliplatin-resistant esophageal cancer cell lines (EC109 and TE-1), making these valuable models for investigating chemoresistance mechanisms .

• RNA modification analysis integration: Given ALYREF's role as an m5C reader protein, experimental designs should incorporate RNA modification analysis techniques:

  Technique	Application	Notes
  m5C-RIP-seq	Global mapping of m5C-modified transcripts bound by ALYREF	Requires specialized library preparation
  Bisulfite sequencing	Site-specific quantification of m5C levels	RNA bisulfite requires modified protocols compared to DNA
  ALYREF-RNA binding assays	Testing direct binding to modified vs. unmodified transcripts	In vitro transcribed RNA with enzymatic modification

• Transcript stability assessment: To evaluate ALYREF's impact on target mRNA stability, implement actinomycin D chase experiments comparing degradation rates in ALYREF-depleted vs. control cells. Research has shown that ALYREF stabilizes TBL1XR1 and KMT2E mRNAs in oxaliplatin-resistant cancer cells .

• Chemoresistance mechanism investigation: When studying ALYREF's role in chemoresistance, include the following experimental approaches:

  • Cell viability assays with dose-response curves to quantify resistance

  • Rescue experiments to establish causality (e.g., ALYREF depletion followed by target mRNA overexpression)

  • In vivo xenograft models with ALYREF modulation to validate in vitro findings

• RNA-protein complex isolation: Implement RNA-protein complex isolation techniques such as formaldehyde cross-linking followed by RIP or CLIP (cross-linking immunoprecipitation) to identify direct RNA targets of ALYREF in cancer contexts.

Recent research has established an ALYREF-TBL1XR1/KMT2E-APOC1 regulatory axis in chemoresistance, where ALYREF recognizes m5C sites on TBL1XR1 and KMT2E mRNAs, stabilizes these transcripts, and promotes APOC1 expression, ultimately contributing to oxaliplatin resistance in esophageal cancer . This mechanistic pathway provides a framework for designing experiments to investigate similar regulatory networks in other cancer types. Researchers should consider this pathway when designing ALYREF-targeted studies in cancer contexts, particularly those involving chemoresistance mechanisms.

What technical challenges might arise when using biotin-conjugated ALYREF antibodies in tissues with high endogenous biotin?

Using biotin-conjugated antibodies in tissues with high endogenous biotin (such as liver, kidney, and brain) presents specific technical challenges requiring methodological adaptations. The following approaches address these issues:

• Endogenous biotin blocking: Implement a comprehensive biotin blocking protocol prior to antibody incubation:

  • Pre-treat sections with avidin (0.1-1 mg/ml) for 15 minutes

  • Rinse thoroughly with PBS

  • Follow with biotin solution (0.1-1 mg/ml) for 15 minutes

  • Rinse thoroughly before proceeding with primary antibody

• Alternative detection systems: Consider using a biotin-free detection system after primary antibody incubation:

  • Polymer-based detection systems (EnVision™, ImmPRESS™)

  • Direct fluorophore-conjugated secondary antibodies

  • Nanobody-based detection systems

• Tissue-specific pre-treatment optimization: For particularly biotin-rich tissues, implement tissue-specific pre-treatments:

  • Liver: Extended permeabilization with 0.3% Triton X-100

  • Brain: Antigen retrieval with formic acid (70%, 10 minutes) followed by standard thermal retrieval

  • Kidney: Extended blocking with 10% normal serum

• Signal validation: Implement parallel validation approaches to confirm specificity:

  • Serial sections with unconjugated ALYREF antibody and conventional detection

  • Peptide competition assays to confirm signal specificity

  • Negative control sections with biotin-conjugated isotype-matched non-specific antibodies

• Endogenous biotin assessment: Include control sections with no primary antibody but with streptavidin-conjugated detection reagent to quantify endogenous biotin levels.

When working with tissues or cells known to have high biotin content, researchers should carefully evaluate whether the advantages of biotin-conjugated antibodies (signal amplification, flexibility in detection systems) outweigh the additional methodological steps required to block endogenous biotin. For ALYREF detection specifically, the protein's predominantly nuclear localization provides a characteristic staining pattern that helps distinguish specific from non-specific signals, as endogenous biotin typically presents with a different subcellular distribution pattern.

How is ALYREF's role in RNA modification reader functionality advancing our understanding of post-transcriptional regulation?

ALYREF's emerging role as a reader of RNA modifications, particularly 5-methylcytosine (m5C), represents a significant advancement in understanding post-transcriptional regulatory mechanisms. This functionality has important implications for experimental design and data interpretation:

• m5C-dependent RNA binding: Recent research has established that ALYREF specifically recognizes and binds m5C-modified mRNAs, including TBL1XR1 and KMT2E transcripts . This preferential binding suggests a mechanism for selective regulation of specific transcripts based on their modification status.

• Integrated epitranscriptome analysis: When investigating ALYREF function, researchers should consider implementing integrated approaches that combine:

  • Transcriptome-wide mapping of m5C sites (RNA bisulfite sequencing, miCLIP)

  • ALYREF binding site identification (CLIP-seq, RIP-seq)

  • Functional outcome assessment (RNA stability, translation efficiency)

• Mechanistic correlation with RNA methyltransferases: Experimental designs should consider the relationship between ALYREF binding and the activity of RNA methyltransferases (particularly NSUN family members) that deposit m5C modifications. This includes:

  • Co-expression analysis in tissue/cell models

  • Sequential ChIP/RIP experiments to identify co-regulated targets

  • Methyltransferase inhibition/depletion effects on ALYREF binding

• Structural biology approaches: Understanding the structural basis of m5C recognition by ALYREF represents an important research direction, potentially employing:

  • Crystallography or cryo-EM of ALYREF bound to modified RNA

  • Mutagenesis of putative m5C-binding domains

  • Molecular dynamics simulations of binding interactions

The discovery that ALYREF functions as an m5C reader has significant implications for various biological processes, including mRNA export, splicing regulation, and transcript stability. In cancer research specifically, this function appears central to chemoresistance mechanisms, as ALYREF-mediated stabilization of TBL1XR1 and KMT2E transcripts promotes APOC1 expression and oxaliplatin resistance in esophageal cancer . Researchers investigating other cancer types or therapeutic resistance mechanisms should consider examining this pathway, integrating m5C mapping with ALYREF binding analysis to identify potential regulatory targets.

How can ALYREF antibodies be utilized in investigating the TREX complex assembly and function?

ALYREF antibodies provide valuable tools for investigating TREX (TRanscription-EXport) complex assembly and function, with several specialized experimental approaches:

• Sequential immunoprecipitation for complex composition analysis: To determine the composition of ALYREF-containing TREX subcomplexes:

  • First IP: Isolate ALYREF-containing complexes using anti-ALYREF antibodies

  • Elution: Mild elution conditions to preserve complex integrity

  • Second IP: Target known TREX components (UAP56/DDX39B, THOC1-7)

  • Analysis: Mass spectrometry to identify differential complex compositions

• Chromatin immunoprecipitation (ChIP) strategies: To investigate co-transcriptional recruitment of ALYREF:

  • Standard ChIP: Mapping ALYREF association with specific genomic regions

  • ChIP-reChIP: Sequential immunoprecipitation with ALYREF antibodies followed by RNA Pol II or other transcription factors

  • ChIP-seq: Genome-wide mapping of ALYREF recruitment patterns

• Proximity-dependent biotin identification (BioID/TurboID): To identify transient or weak interactions within the TREX complex:

  • Generate ALYREF-BioID fusion proteins

  • Express in cells of interest with biotin supplementation

  • Isolate biotinylated proteins and identify by mass spectrometry

  • Compare with published TREX component lists

• Dynamic assembly visualization: To observe TREX complex formation in real-time:

  • Fluorescence recovery after photobleaching (FRAP) of fluorescently tagged ALYREF

  • Single-molecule tracking using ALYREF antibody fragments

  • Förster resonance energy transfer (FRET) between labeled ALYREF and other TREX components

• Functional perturbation analysis: To assess the impact of ALYREF on TREX function:

  • mRNA export assays following ALYREF depletion or mutation

  • Pulse-chase experiments to measure mRNA export kinetics

  • Single molecule RNA visualization with combined ALYREF immunostaining

Recent research has demonstrated that ALYREF's interactions within the TREX complex can be modulated by its binding to m5C-modified RNAs . This suggests that RNA modifications might influence TREX assembly or function, representing an important area for future investigation. Researchers should consider how RNA modification status might affect ALYREF's interactions with other TREX components when designing experiments to study this complex. Additionally, the role of ALYREF in TREX function may be particularly important in cancer contexts, where altered RNA processing and export contribute to oncogenic mechanisms.

What are the most critical considerations when selecting and validating ALYREF antibodies for specific research applications?

Selecting and validating ALYREF antibodies requires systematic consideration of multiple factors to ensure experimental reliability and reproducibility. The following framework provides a comprehensive approach:

• Application-specific antibody selection: Different experimental applications have distinct antibody requirements:

  • For Western blotting: Select antibodies validated against denatured epitopes

  • For immunoprecipitation: Choose antibodies recognizing native conformations

  • For immunohistochemistry: Verify compatibility with fixation methods

  • For multiplexed applications: Consider conjugated formats (including biotin) with minimal interference

• Cross-reactivity assessment: Evaluate species cross-reactivity based on conservation analysis and empirical validation. Current evidence indicates that many ALYREF antibodies recognize human, mouse, rat, and pig orthologs, but this should be verified for each application .

• Epitope location considerations: ALYREF contains distinct functional domains, and antibodies targeting different regions may yield varying results:

  • N-terminal (aa 1-100): Contains RNA recognition motif, important for RNA binding

  • Middle region (aa 100-200): Includes nucleocytoplasmic shuttling signals

  • C-terminal (aa 200-257): Contains protein-protein interaction domains

• Biotin conjugation impact assessment: For biotin-conjugated antibodies specifically:

  • Verify that conjugation does not affect epitope recognition

  • Implement appropriate blocking for endogenous biotin

  • Consider detection system compatibility with experimental design

• Validation hierarchy implementation: Implement a multi-level validation strategy:

  • Level 1: Basic validation (expected molecular weight, cellular localization)

  • Level 2: Genetic validation (siRNA knockdown, CRISPR knockout)

  • Level 3: Independent antibody correlation (different epitopes/clones)

  • Level 4: Functional validation (expected interaction partners or dynamics)

• Context-specific validation: Validate antibody performance in the specific experimental context:

  • Cell/tissue type of interest

  • Disease state (normal vs. pathological)

  • Treatment conditions

  • Fixation/preservation methods

The importance of rigorous validation cannot be overstated, particularly given ALYREF's critical roles in fundamental cellular processes and emerging significance in cancer mechanisms. Recent research highlighting ALYREF's role in chemoresistance through RNA modification-dependent mechanisms underscores the need for reliable antibody tools to further investigate these pathways . Researchers should prioritize antibodies with comprehensive validation data across multiple applications and implement their own validation protocols specific to their experimental systems.