IFRD1 Antibody，Biotin conjugated

What is IFRD1 and why is it a significant research target?

IFRD1 (Interferon-Related Developmental Regulator 1) functions as a transcriptional modulator implicated in multiple cellular processes. Research significance stems from its roles in:

• Promoting tumor cell survival under glutamine starvation by inhibiting autophagy through ATG14 degradation

• Regulating gene activity in proliferative and/or differentiative pathways induced by nerve growth factor (NGF)

• Functioning as a negative regulator of thermogenic and mitochondrial gene expression in brown adipocytes

• Suppressing bone formation through modulation of NF-κB activity in osteoblasts

The biotin-conjugated antibody provides enhanced sensitivity for detecting this multifunctional protein across different experimental contexts.

What are the recommended applications for biotin-conjugated IFRD1 antibody?

Biotin-conjugated IFRD1 antibody is particularly suitable for:

• ELISA (enzyme-linked immunosorbent assay) - Primary application with validated specificity

• Immunohistochemistry - For tissue localization studies with signal amplification

• Flow cytometry - Leveraging biotin-streptavidin detection systems

• Immunoprecipitation experiments requiring signal enhancement

The biotin conjugation enables amplified detection through secondary streptavidin systems, making it valuable for detecting low-abundance IFRD1 in complex samples. For other applications like Western blotting, unconjugated antibodies may be preferable based on experimental design requirements .

How can biotin-conjugated IFRD1 antibody be optimized for studying glutamine metabolism in cancer cells?

For investigating IFRD1's role in glutamine metabolism:

• Dual immunofluorescence approach: Combine biotin-conjugated IFRD1 antibody with antibodies against ATG14 or TRIM21 to visualize their interaction during glutamine starvation

• Sequential detection protocol:

  • First detect IFRD1 using biotin-conjugated antibody with streptavidin-HRP

  • Follow with antibodies against autophagy markers

  • Measure co-localization under normal and glutamine-deprived conditions

• Quantitative analysis workflow:

  • Culture cells in glutamine-depleted media or treat with CB-839 (glutaminase inhibitor)

  • Fix cells at different time points

  • Process for immunodetection with biotin-conjugated IFRD1 antibody

  • Quantify intensity changes relative to control conditions

This approach allows correlation of IFRD1 expression levels with autophagy inhibition during metabolic stress in cancer models .

What methodological approaches are recommended for studying IFRD1's interaction with transcriptional complexes?

To investigate IFRD1's role in transcriptional regulation:

• Chromatin immunoprecipitation (ChIP) protocol adaptation:

  • Use biotin-conjugated IFRD1 antibody with streptavidin magnetic beads

  • Isolate protein-DNA complexes

  • Analyze binding to promoter regions of target genes (e.g., PGC-1α, Smad7)

• Co-immunoprecipitation strategy:

  • Precipitate IFRD1 complexes using biotin-conjugated antibody

  • Probe for interaction partners including:

    • mSIN3B and HDAC complexes (for thermogenic gene regulation)

    • NF-κB p65 (for bone formation regulation)

    • Sp1 transcription factor complexes

• Sequential ChIP approach:

  • First ChIP with biotin-IFRD1 antibody

  • Second ChIP with antibodies against suspected interaction partners

  • Identify co-occupied genomic regions

These methodologies help elucidate how IFRD1 coordinates with histone deacetylases to modulate gene expression in different tissue contexts .

How can IFRD1's role in stress responses be effectively studied using biotin-conjugated antibodies?

For investigating stress-induced IFRD1 regulation:

• Temporal expression analysis protocol:

  • Treat cells with stress inducers (tunicamycin, nutrient deprivation)

  • Collect samples at multiple timepoints (2h, 4h, 6h, 8h)

  • Use biotin-conjugated IFRD1 antibody for ELISA or IHC detection

  • Compare with primary transcript measurement by RT-PCR

• Subcellular fractionation workflow:

  • Separate nuclear and cytoplasmic fractions after stress induction

  • Detect IFRD1 localization changes using biotin-conjugated antibody

  • Correlate with phosphorylation status of eIF2α

• Reporter gene assay strategy:

  • Create constructs containing IFRD1 upstream ORF regions

  • Monitor translation regulation during stress

  • Use biotinylated IFRD1 antibody to track endogenous protein production

This integrated approach reveals both transcriptional and post-transcriptional regulation of IFRD1 during cellular stress responses .

How can background signal be minimized when using biotin-conjugated IFRD1 antibody in tissues with high endogenous biotin?

To reduce background from endogenous biotin when using biotin-conjugated IFRD1 antibody:

• Pre-blocking protocol:

  • Implement avidin/biotin blocking step (sequential incubation with avidin followed by biotin)

  • Use commercial biotin blocking systems before primary antibody incubation

  • Include 0.1% non-fat dry milk in blocking buffer

• Sample preparation optimization:

  • For tissues with high endogenous biotin (liver, kidney, brain):

    • Reduce fixation time to minimize epitope masking

    • Test different antigen retrieval methods (citrate vs. EDTA buffers)

    • Implement streptavidin-based detection systems with lower background

• Control implementations:

  • Always include a negative control omitting primary antibody

  • Use tissue from IFRD1 knockout mice (when available) as specificity control

  • Include isotype control at the same concentration as primary antibody

These approaches significantly improve signal-to-noise ratio in tissues with high endogenous biotin content, particularly brain samples where IFRD1 expression has been documented .

What are the recommended approaches for validating IFRD1 antibody specificity in various experimental systems?

To validate biotin-conjugated IFRD1 antibody specificity:

• Multi-method validation workflow:

  • Compare staining patterns with at least two independent IFRD1 antibodies

  • Perform RNA interference (siRNA against IFRD1) followed by antibody detection

  • Use recombinant IFRD1 protein for pre-absorption tests

• Expression correlation analysis:

  • Parallel analysis of protein detection and mRNA expression

  • Compare antibody staining intensity with RT-PCR quantification

  • Confirm increased expression during known induction conditions (e.g., stress, adrenergic stimulation)

• Molecular weight verification protocol:

  • Run western blot with unconjugated antibody alongside biotin-conjugated version

  • Confirm 50 kDa band (predicted size for IFRD1)

  • Check for absence of non-specific bands

These validation steps ensure experimental results reflect genuine IFRD1 biology rather than non-specific antibody interactions.

How should storage and handling of biotin-conjugated IFRD1 antibody be managed to maintain optimal activity?

For maintaining optimal activity of biotin-conjugated IFRD1 antibody:

• Storage protocol:

  • Aliquot upon receipt to minimize freeze-thaw cycles

  • Store at -20°C as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles that can lead to biotin degradation

• Working solution preparation:

  • Dilute in buffer containing carrier protein (0.5-1% BSA)

  • For long-term storage of working dilutions, add preservative (0.02-0.05% sodium azide)

  • Prepare fresh dilutions for critical experiments

• Quality control monitoring:

  • Implement routine functional testing after storage periods

  • Use consistent positive control samples to monitor performance over time

  • Document lot-to-lot variation if multiple purchases are necessary

Following these guidelines maximizes antibody stability while ensuring reproducible experimental results across extended research projects.

What experimental design is optimal for studying IFRD1's role in cancer metabolism using biotin-conjugated antibodies?

For investigating IFRD1 in cancer metabolism:

• Cell line selection strategy:

  • Compare hepatocellular carcinoma (HCC) cells (where IFRD1's role is documented)

  • Include non-transformed hepatocytes as controls

  • Consider glutamine-dependent vs. non-dependent cancer lines

• Treatment protocol design:

  • Implement glutamine starvation time course (2h, 4h, 8h, 12h, 24h)

  • Include CB-839 (glutaminase inhibitor) conditions

  • Add IFRD1 knockdown/overexpression experimental groups

• Multiparameter analysis workflow:

  • Use biotin-conjugated IFRD1 antibody for protein detection

  • Simultaneously measure ATG14 levels and autophagy markers

  • Correlate with cell viability and metabolic parameters

• In vivo experimental approach:

  • Establish xenograft models with IFRD1-modulated cancer cells

  • Perform IHC with biotin-conjugated IFRD1 antibody

  • Correlate IFRD1 expression with tumor growth/metabolism markers

This comprehensive approach allows for mechanistic understanding of how IFRD1 promotes tumor cell survival under metabolic stress conditions .

How can biotin-conjugated IFRD1 antibody be incorporated into multiplexed imaging protocols?

For multiplexed imaging with biotin-conjugated IFRD1 antibody:

• Sequential multiplexing protocol:

  • First round: Detect IFRD1 using biotin-conjugated antibody with streptavidin-fluorophore

  • Strip or quench signal

  • Subsequent rounds: Detect additional targets

  • Use image registration software to align multiple rounds

• Spectral unmixing approach:

  • Select fluorophores with minimal spectral overlap

  • Use streptavidin conjugated to spectrally distinct fluorophores for IFRD1 detection

  • Implement computational spectral unmixing for clean signal separation

• Tyramide signal amplification integration:

  • Use biotin-conjugated IFRD1 antibody with HRP-streptavidin

  • Develop with tyramide-fluorophore for covalent signal deposition

  • Heat-strip antibodies while preserving amplified signal

  • Proceed with subsequent markers

These protocols enable visualization of IFRD1 alongside interaction partners and relevant signaling molecules in complex tissue samples.

What considerations are important when designing studies to investigate IFRD1's post-transcriptional regulation using biotin-conjugated antibodies?

For studying IFRD1's post-transcriptional regulation:

• Combined protein-RNA detection strategy:

  • Implement RNA-FISH for IFRD1 transcript visualization

  • Follow with biotin-conjugated IFRD1 antibody immunodetection

  • Correlate mRNA and protein localization under various stress conditions

• Translation inhibition experimental design:

  • Treat cells with cycloheximide or puromycin

  • Monitor IFRD1 protein stability using biotin-conjugated antibody

  • Compare with actinomycin D treatments to distinguish transcriptional vs. post-transcriptional effects

• uORF analysis methodology:

  • Create reporter constructs with IFRD1's upstream open reading frame

  • Induce cellular stress (tunicamycin treatment)

  • Monitor reporter expression alongside endogenous IFRD1 using biotin-conjugated antibody

• eIF2α phosphorylation correlation analysis:

  • Simultaneously detect phospho-eIF2α and IFRD1

  • Track temporal relationship during stress response

  • Implement eIF2α phosphorylation inhibitors to confirm mechanism

This multifaceted approach reveals how post-transcriptional mechanisms regulate IFRD1 expression during cellular stress responses and how this impacts downstream cellular functions.