ALYREF Antibody, HRP conjugated

What is ALYREF and what are its key functions in cellular processes?

ALYREF (Aly/REF export factor, also known as THOC4) is a heat-stable, nuclear protein that functions as a molecular chaperone. It plays crucial roles in multiple cellular processes including:

• RNA metabolism and nuclear export of mRNAs

• Formation of the TREX complex with RNA helicase UAP56 and the THO sub-complex

• Regulation of mRNA stability and gene transcription

• Dimerization and DNA binding of basic region-leucine zipper (bZIP) proteins

Within cells, ALYREF localizes to the nucleus, nuclear speckles, and occasionally the cytoplasm. It colocalizes with the core EJC, THOC4, NXF1, and DDX39B in nuclear speckles and travels to the cytoplasm as part of the exon junction complex (EJC) bound to mRNA .

What advantages does an HRP-conjugated ALYREF antibody offer compared to unconjugated versions?

HRP-conjugated ALYREF antibodies provide several methodological advantages over unconjugated antibodies:

• Direct detection without the need for secondary antibodies, reducing protocol time by 1-2 hours

• Elimination of background signal that can arise from cross-reactivity of secondary antibodies

• Enhanced sensitivity due to direct enzymatic signal amplification

• Reduced variability between experiments as the conjugation ratio is standardized

• Simplified multiplex staining with antibodies from the same host species

The HRP conjugation leverages Lightning-Link® conjugation technology to deliver highly consistent recombinant conjugates, providing researchers with reliable antibody-label combinations for detection systems .

What applications are HRP-conjugated ALYREF antibodies validated for?

According to commercial validation data, HRP-conjugated ALYREF antibodies are primarily validated for:

• Western Blotting (WB): Detecting ALYREF protein (~27-30 kDa) in cell and tissue lysates

• ELISA: For quantitative detection in solution-based immunoassays

While the HRP-conjugated versions are specifically validated for these applications, it's worth noting that unconjugated ALYREF antibodies have broader application validation including Immunohistochemistry (IHC), Immunofluorescence (IF), and Flow Cytometry .

How should I optimize Western blot protocols when using HRP-conjugated ALYREF antibodies?

For optimal Western blot results with HRP-conjugated ALYREF antibodies:

Sample Preparation:

• Include phosphatase inhibitors in lysis buffer as ALYREF function can be regulated by phosphorylation

• Load 20-40 μg of total protein per lane

• Use fresh samples where possible, as ALYREF may degrade in long-term storage

Protocol Optimization:

• Blocking: Use 5% non-fat milk in TBST (avoid BSA which may increase background)

• Dilution: Start with 1:1000 dilution and adjust based on signal strength

• Incubation: 1-2 hours at room temperature or overnight at 4°C

• Washing: 5 × 5 minute washes with TBST to reduce background

• Detection: Use ECL substrate optimized for moderate-abundance proteins

• Expected band size: ~30 kDa (observed) although the calculated MW is 26 kDa

The discrepancy between calculated (26 kDa) and observed (30 kDa) molecular weight is due to post-translational modifications of ALYREF in cells, which affects its mobility during electrophoresis .

What controls should be included when using HRP-conjugated ALYREF antibodies in experimental workflows?

Robust experimental design requires several controls:

Positive Controls:

• HeLa, A549, B-cells, SKOV3, and HL-60 cell lysates have been verified to express detectable levels of ALYREF

• Mouse spleen and rat brain tissue lysates for rodent experiments

Negative Controls:

• Primary antibody omission control

• Isotype control antibody (rabbit IgG-HRP at matching concentration)

• ALYREF-depleted samples through siRNA knockdown or CRISPR-Cas9

Loading Controls:

• Use GAPDH, β-actin, or α-tubulin for cytoplasmic normalization

• Use Lamin A/C or Histone H3 when analyzing nuclear fractions where ALYREF predominantly localizes

Additional Validation:

• If studying specific binding interactions, include competitive blocking with recombinant ALYREF protein

• For subcellular localization studies, use nuclear/cytoplasmic fractionation to confirm compartment-specific signals

How should HRP-conjugated ALYREF antibodies be stored and handled to maintain optimal performance?

Proper storage and handling are critical for maintaining antibody performance:

Storage Recommendations:

• Store at -20°C in small aliquots (10-20 μL) to avoid repeated freeze-thaw cycles

• Most products are supplied in a buffer containing glycerol (50%), which prevents freezing at -20°C

• Valid for approximately 12 months when properly stored

Handling Guidelines:

• Thaw aliquots at room temperature and briefly centrifuge before opening

• Never vortex the antibody solution (gentle inversion is sufficient)

• Always keep on ice when in use

• Return to -20°C immediately after use

• Avoid more than 5 freeze-thaw cycles, which can reduce activity by up to 50%

• Protect from light, as HRP is light-sensitive

• Never use sodium azide, which inhibits HRP activity

How can HRP-conjugated ALYREF antibodies be used to investigate cancer-related pathways based on recent findings?

Recent research shows ALYREF plays significant roles in cancer progression through several mechanisms:

Cancer Signaling Pathway Analysis:

• EGFR-STAT3 Axis: ALYREF stabilizes EGFR mRNA by binding to its m5C-modified sites, activating STAT3 signaling. Researchers can use HRP-conjugated ALYREF antibodies in chromatin immunoprecipitation (ChIP) assays followed by Western blot to detect this interaction .

• MYC Feedback Loop: ALYREF forms a positive feedback loop with MYC in glioblastoma. MYC regulates ALYREF transcription, while ALYREF stabilizes MYC mRNA by binding to its 3'-UTR. This can be investigated using RNA immunoprecipitation followed by western blotting with HRP-conjugated ALYREF antibodies .

• mTOR Pathway Components: ALYREF promotes colorectal cancer by regulating RPS6KB2 and RPTOR transcripts. Researchers can use HRP-conjugated ALYREF antibodies in co-IP experiments to study the ALYREF-ELAVL1 complex formation that facilitates m5C recognition and nuclear export .

Experimental Approach:

• Use HRP-conjugated ALYREF antibodies for Western blot analysis of tumor vs. normal tissues

• Compare ALYREF expression with clinical parameters like tumor stage, grade, and patient survival

• Combine with m5C RNA immunoprecipitation to identify ALYREF-regulated transcripts

How can I design experiments to study ALYREF's role in RNA m5C modification using HRP-conjugated antibodies?

ALYREF functions as an m5C reader protein that recognizes 5-methylcytosine-modified RNAs. To study this function:

Experimental Design:

• RNA-Protein Interaction Studies:

  • RNA immunoprecipitation (RIP) with ALYREF antibody followed by bisulfite sequencing

  • Western blot with HRP-conjugated ALYREF antibody to confirm successful pulldown

  • Analyze enrichment of specific mRNA targets (e.g., EGFR, MYC, RPS6KB2, RPTOR)

• Protein Complex Analysis:

  • Co-immunoprecipitation to identify ALYREF binding partners (such as ELAVL1)

  • Use HRP-conjugated ALYREF antibodies for Western blot detection

  • Analyze formation of ALYREF-containing complexes in response to cellular stress or cancer development

• Functional Validation:

  • Perform luciferase reporter assays with wild-type and m5C-mutated 3'UTR constructs

  • Use ALYREF knockdown/overexpression followed by Western blot with HRP-conjugated antibodies

  • Measure changes in target mRNA stability using actinomycin D chase experiments

Data Interpretation:

• Compare m5C levels in target mRNAs with ALYREF binding efficiency

• Correlate with mRNA stability, export, and protein expression

• Analyze the functional consequences through cell proliferation, migration, and invasion assays

What is the significance of ALYREF subcellular localization and how can HRP-conjugated antibodies help track its dynamic movement?

ALYREF exhibits complex subcellular dynamics critical to its function:

Subcellular Distribution Pattern:

• Primarily nuclear with enrichment in nuclear speckles

• Colocalizes with THOC4, NXF1, and DDX39B in the nucleus

• Shuttles to the cytoplasm as part of the exon junction complex (EJC) bound to mRNA

Experimental Approaches:

• Subcellular Fractionation:

  • Separate nuclear, nucleolar, and cytoplasmic fractions

  • Use HRP-conjugated ALYREF antibodies for Western blot analysis of each fraction

  • Compare distribution under normal vs. stress conditions or in cancer vs. normal cells

• Proximity-Based Protein Interaction Studies:

  • Perform proximity ligation assays (PLA) with ALYREF and known partners

  • Use HRP-conjugated antibodies for detection in fixed cells

  • Quantify interactions in different subcellular compartments

• Temporal Dynamics:

  • Track ALYREF localization during cell cycle progression

  • Monitor redistribution after transcriptional inhibition or RNA export blockade

  • Use HRP-conjugated antibodies for time-course Western blot analysis

This approach can reveal how ALYREF's distribution changes during cancer progression, particularly when the nuclear export of specific oncogenic mRNAs is altered.

What are the common issues in Western blotting with HRP-conjugated ALYREF antibodies and how can they be resolved?

Researchers commonly encounter several challenges when using HRP-conjugated ALYREF antibodies:

Problem: Multiple bands detected

• Cause: ALYREF undergoes post-translational modifications; proteolytic degradation

• Solution: Use fresh samples with protease inhibitors; verify with positive control lysates from HeLa or A549 cells; compare with literature reporting ~30 kDa observed vs. 26 kDa calculated size

Problem: Weak or no signal

• Cause: Insufficient protein, antibody degradation, inefficient transfer

• Solution: Increase protein loading (40-60 μg); reduce antibody dilution to 1:500; optimize transfer conditions; ensure proper storage of antibody; check HRP activity with substrate directly on membrane

Problem: High background

• Cause: Insufficient blocking, contaminated TBST, excessive antibody

• Solution: Increase blocking time (overnight at 4°C); prepare fresh TBST; increase washing steps (5 × 10 min); optimize antibody dilution; use specialized blocking buffers for HRP-conjugated antibodies

Problem: Inconsistent results between experiments

• Cause: Antibody degradation, variable lysate preparation, inconsistent transfer

• Solution: Use single-use aliquots; standardize lysate preparation protocol; include consistent positive controls; use internal loading controls for normalization

How can I differentiate between specific and non-specific signals when using HRP-conjugated ALYREF antibodies?

Distinguishing specific from non-specific signals requires systematic validation:

Validation Approaches:

• Genetic Validation:

  • Perform siRNA or shRNA knockdown of ALYREF

  • Compare Western blot signals between control and knockdown samples

  • Specific bands should decrease significantly in knockdown samples

• Molecular Weight Analysis:

  • ALYREF shows apparent molecular weight of ~30 kDa (though calculated MW is 26 kDa)

  • Non-specific bands that don't change with knockdown should be considered artifacts

  • Consistent bands across different cell types increase confidence in specificity

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel Western blots with competed and non-competed antibody

  • Specific signals should disappear in the competed lane

• Alternative Antibody Validation:

  • Compare results with a different ALYREF antibody (e.g., clone EPR17942 vs. 11G5)

  • Specific signals should be consistent between different antibody clones

Interpretation Table:

What are the critical parameters for optimizing immunoprecipitation using HRP-conjugated ALYREF antibodies?

While HRP-conjugated antibodies are primarily designed for detection rather than immunoprecipitation, they can be useful for analyzing IP samples. Here are critical parameters:

For Detection of IP Samples:

• Sample Preparation:

  • For IP, use unconjugated ALYREF antibodies (HRP conjugation may hinder antigen binding in solution)

  • Use HRP-conjugated antibodies only for Western blot detection after IP

  • Include heavy chain blocking reagents to prevent detection of IgG (~50 kDa) which can mask nearby proteins

• Protocol Modifications:

  • Use protein G magnetic beads for rabbit antibodies, protein A for mouse antibodies

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Gentle elution conditions to maintain antibody integrity

  • Include RNase inhibitors when studying RNA-protein interactions

• Controls:

  • Input control (10% of starting material)

  • IgG control (same species as primary antibody)

  • No-antibody control (beads only)

  • Positive control (known ALYREF interacting protein like UAP56/DDX39B)

• Special Considerations for RNA-Protein Interaction Studies:

  • Use UV cross-linking to stabilize RNA-protein interactions

  • Include RNase inhibitors in all buffers

  • Consider formaldehyde cross-linking for protein complex studies

  • Validate RNA enrichment using qRT-PCR for known targets (EGFR, MYC)

How do ALYREF expression levels correlate with cancer progression and what implications does this have for research?

ALYREF expression shows significant correlation with cancer progression across multiple studies:

Expression Patterns in Cancer:

• Liver Hepatocellular Carcinoma (LIHC): Higher ALYREF expression correlates with advanced tumor classification, TNM stage, and larger tumor size

• Glioblastoma (GBM): ALYREF is frequently increased in GBM tissues

• Colorectal Cancer (CRC): ALYREF overexpression predicts poor prognosis

Clinical Correlations:

Research Applications:

• Biomarker Development:

  • Use HRP-conjugated ALYREF antibodies for tissue microarray analysis

  • Correlate expression with clinical parameters and patient outcomes

  • Develop standardized scoring systems for ALYREF immunostaining

• Therapeutic Target Identification:

  • Screen for compounds that modulate ALYREF expression or function

  • Analyze effects on cancer cell proliferation, migration, and invasion

  • Evaluate combinations with standard therapies

• Mechanistic Studies:

  • Investigate ALYREF's role in different cancer types

  • Compare expression with other cancer-related proteins (EGFR, MYC, STAT3)

  • Study downstream effects on RNA metabolism and gene expression

How can I use HRP-conjugated ALYREF antibodies to investigate the relationship between ALYREF and specific signaling pathways in cancer?

ALYREF interacts with multiple cancer-related signaling pathways, which can be investigated using HRP-conjugated antibodies:

EGFR-STAT3 Signaling Axis:

• Experimental Approach:

  • Treat cells with EGFR inhibitors (e.g., erlotinib) or STAT3 inhibitors

  • Analyze ALYREF expression and localization by Western blot

  • Perform siRNA knockdown of ALYREF and measure EGFR and p-STAT3 levels

  • Use HRP-conjugated ALYREF antibodies for detection in Western blot analysis

• Expected Outcomes:

  • ALYREF knockdown should decrease EGFR expression at mRNA and protein levels

  • EGFR inhibition may not affect ALYREF levels but should block its downstream effects

  • Changes in STAT3 phosphorylation without changes in total STAT3

MYC Feedback Loop:

• Experimental Approach:

  • Compare MYC and ALYREF levels across cancer cell lines

  • Perform MYC knockdown and measure ALYREF expression

  • Test ALYREF knockdown effect on MYC stability using cycloheximide chase assay

  • Detect proteins using HRP-conjugated antibodies

• Expected Outcomes:

  • Positive correlation between MYC and ALYREF expression

  • MYC knockdown should decrease ALYREF transcription

  • ALYREF knockdown should decrease MYC mRNA stability and protein levels

  • Breaking this feedback loop should reduce cancer cell proliferation

mTOR Pathway:

• Experimental Approach:

  • Use rapamycin to inhibit mTOR signaling and analyze ALYREF function

  • Study RPS6KB2 and RPTOR mRNA export in ALYREF knockdown cells

  • Analyze protein synthesis rates with ALYREF manipulation

  • Use HRP-conjugated antibodies for Western blot detection

• Expected Outcomes:

  • ALYREF should affect nuclear export of specific mTOR pathway component mRNAs

  • Changes in protein synthesis rates in ALYREF-manipulated cells

  • Altered response to mTOR inhibitors with ALYREF knockdown

What is the relationship between ALYREF and RNA modifications in cancer, and how can this be studied?

ALYREF functions as an m5C reader protein with important implications for cancer research:

ALYREF and RNA m5C Modification:

• ALYREF recognizes m5C-modified RNA sites, particularly in 3'UTR regions

• This recognition stabilizes target mRNAs and enhances their expression

• Key targets include EGFR, MYC, RPS6KB2, and RPTOR mRNAs

Comprehensive Research Strategy:

• Transcriptome-wide Identification of ALYREF m5C Targets:

  • Perform ALYREF RNA immunoprecipitation followed by bisulfite sequencing (RIP-BS-seq)

  • Use HRP-conjugated ALYREF antibodies to confirm protein levels

  • Identify common sequence or structural motifs in bound RNAs

• Mechanistic Validation:

  • Measure m5C levels in target mRNAs using m5C-specific antibodies

  • Create m5C site mutations in target 3'UTRs and analyze ALYREF binding

  • Study mRNA half-life with/without ALYREF expression

  • Compare nuclear vs. cytoplasmic distribution of target mRNAs

• Functional Consequences:

  • Correlate m5C levels with ALYREF binding efficiency and mRNA stability

  • Analyze protein expression of m5C-modified targets in cancer vs. normal tissues

  • Evaluate the effect of disrupting this pathway on cancer phenotypes

  • Use HRP-conjugated ALYREF antibodies for protein detection in these assays

Data Interpretation Framework:

• Higher m5C levels should correlate with increased ALYREF binding

• ALYREF binding should correlate with increased mRNA stability and protein expression

• Disrupting ALYREF-m5C interaction should destabilize target mRNAs and reduce protein levels

• Cancer tissues likely show higher levels of both m5C modification and ALYREF expression