IFRD1 Antibody，FITC conjugated

What is IFRD1 and why is it significant for research?

IFRD1 (Interferon-related developmental regulator 1), also known as Nerve growth factor-inducible protein PC4, is a multifunctional protein that plays crucial roles in various biological processes. It regulates gene activity in proliferative and differentiative pathways induced by NGF (Nerve Growth Factor) and may function as an autocrine factor that attenuates or amplifies initial ligand-induced signals . Research significance stems from IFRD1's involvement in diverse physiological and pathological processes, including:

• Immune evasion mechanisms exploited by human papillomavirus through EGFR-IFRD1-mediated pathways

• Skeletal muscle regeneration and regulation of osteoclast differentiation

• Maintenance of bladder epithelial homeostasis with implications for urological disorders

• Tumor cell survival under glutamine starvation conditions in hepatocellular carcinoma

The wide-ranging functions of IFRD1 make it an important target for investigating cellular signaling pathways, immune responses, and potential therapeutic interventions.

What detection methods are compatible with FITC-conjugated IFRD1 antibodies?

FITC-conjugated IFRD1 antibodies are optimized for fluorescence-based detection methods. The recommended techniques include:

• Flow Cytometry: Ideal for quantitative analysis of IFRD1 expression in single-cell suspensions. Protocol optimization should include titration experiments to determine optimal antibody concentration and inclusion of appropriate isotype controls.

• Immunofluorescence Microscopy: Suitable for visualizing cellular localization of IFRD1. The fluorescein excitation maximum at 495nm and emission maximum at 519nm provides good signal-to-noise ratio with standard FITC filter sets.

• High-Content Imaging: For automated, quantitative analysis of IFRD1 expression and localization across large cell populations.

• Intracellular Flow Cytometry: Requires permeabilization steps as IFRD1 functions primarily as an intracellular protein regulating differentiation pathways .

When selecting detection methods, researchers should consider that IFRD1 exhibits differential expression patterns across tissues, with notable expression in the superficial urothelial cell layer of the bladder as demonstrated through X-gal staining in IFRD1 reporter mice .

How should I optimize fixation and permeabilization protocols for IFRD1 detection?

Optimization of fixation and permeabilization protocols is critical for successful detection of IFRD1 using FITC-conjugated antibodies. Based on research practices:

Fixation:

• Paraformaldehyde (4%): Recommended for general applications, preserves cellular architecture while maintaining epitope accessibility.

• Cross-linking fixatives containing glutaraldehyde: Effective for preserving IFRD1 detection in whole organ preparations, as demonstrated in bladder tissue analyses .

• Methanol fixation: May be suitable for some applications but can affect FITC fluorescence intensity.

Permeabilization:

• Mild detergents (0.1-0.5% Triton X-100 or 0.1% Saponin): Suitable for intracellular detection of IFRD1.

• Digitonin (10-50 μg/ml): For selective permeabilization of the plasma membrane while preserving internal membranes.

Optimization steps:

• Compare multiple fixation times (10-30 minutes) at room temperature

• Test different permeabilization conditions using a cell type similar to your experimental system

• Include positive controls where IFRD1 is known to be expressed (e.g., stimulated immune cells or urothelial tissue sections)

• Compare results with non-FITC conjugated IFRD1 antibodies to assess potential loss of signal during fixation

Research with IFRD1 antibodies in bladder tissue has demonstrated successful detection using both fresh frozen sections and whole-organ fixed preparations , indicating flexibility in fixation approaches depending on experimental needs.

What are the optimal blocking conditions for reducing background when using FITC-conjugated IFRD1 antibodies?

Background reduction is essential for generating reliable data with FITC-conjugated IFRD1 antibodies. The following blocking strategies are recommended:

Primary blocking agents:

• 5-10% normal serum: Use serum from the species in which the secondary antibody was raised (if using detection systems with secondary antibodies)

• 3-5% BSA in PBS: Effective for most applications

• Commercial blocking buffers: Specifically formulated for fluorescence applications to minimize autofluorescence

Additional blocking considerations:

• Fc receptor blocking: Critical when examining immune cells that express Fc receptors to prevent non-specific binding

• Biotin/avidin blocking: Important if using detection systems involving biotin

• Autofluorescence reduction: Pre-treatment with 0.1-1% sodium borohydride can reduce cellular autofluorescence, particularly relevant in tissues with high collagen content

Optimization protocol:

• Test multiple blocking buffer formulations

• Include isotype control antibodies conjugated to FITC

• Perform blocking time course (30 minutes to overnight at 4°C)

• Include antigen competition controls to verify specificity

The specificity of antibody binding is particularly important when investigating IFRD1's interactions with its numerous binding partners, such as those identified in the human urothelial cell line through co-immunoprecipitation followed by tandem mass spectrometry .

How can I reliably quantify IFRD1 expression levels using FITC-conjugated antibodies?

Reliable quantification of IFRD1 expression requires careful experimental design and appropriate controls. The following approaches are recommended:

Flow cytometry quantification:

• Standard curve calibration: Use calibration beads with known quantities of FITC molecules to establish fluorescence intensity standards

• Mean Fluorescence Intensity (MFI): Calculate relative expression levels by comparing sample MFI to control populations

• Population analysis: Determine percentage of cells expressing IFRD1 above threshold established with isotype controls

Microscopy-based quantification:

• Integrated density measurements: Calculate the product of area and mean gray value

• Background correction: Subtract average background readings from adjacent non-expressing regions

• Nuclear/cytoplasmic ratio analysis: IFRD1 can shuttle between cellular compartments based on activation state

Western blot correlation:
When possible, correlate fluorescence-based measurements with western blot quantification using non-conjugated IFRD1 antibodies. Studies have used antibodies from Abcam (anti-IFRD1) with successful protein detection .

RNA-protein correlation:
Consider parallel quantification of IFRD1 transcript levels using RT-qPCR as performed in bladder homeostasis studies . This can help validate antibody-based protein measurements and identify potential post-transcriptional regulation.

What are the most effective methods for studying IFRD1 interaction partners using FITC-conjugated antibodies?

Studying IFRD1 interaction partners requires specialized approaches that maintain protein-protein interactions while leveraging the fluorescent properties of FITC conjugation:

Proximity Ligation Assay (PLA):

• Use FITC-conjugated anti-IFRD1 antibody with non-conjugated antibodies against suspected interaction partners

• Implement oligonucleotide-linked secondary antibodies that generate fluorescent spots when proteins are in close proximity

• Quantify interaction events as discrete fluorescent signals

Fluorescence Resonance Energy Transfer (FRET):

• Pair FITC-conjugated IFRD1 antibody (donor) with antibodies against interaction partners conjugated to compatible acceptor fluorophores

• Measure energy transfer to determine molecular proximity

• Calculate FRET efficiency to estimate relative distances between proteins

Co-immunoprecipitation with fluorescence detection:
Building on established co-IP methods used for IFRD1 , incorporate fluorescence-based detection:

• Use FITC-conjugated IFRD1 antibodies for immunoprecipitation

• Detect co-precipitated proteins using complementary techniques

• Implement fluorescence scanning of IP products separated by gel electrophoresis

Functional validation approaches:
For validating interactions functionally, consider knockdown approaches using siRNA techniques as documented for IFRD1. Previous research has successfully employed 19-nucleotide siRNA sequences specific to PC4/Tis7 (IFRD1) with sequences:

• PC4/2: 5′-GAGAGCAGATGTTGGAGAA-3′

• PC4/7: 5′-GCGCATGTATATTGATAGC-3′

These approaches can help identify and validate interaction partners such as those involved in IFRD1's roles in mediating EGFR signaling or its interaction with proteins involved in translational processes .

How can I validate the specificity of FITC-conjugated IFRD1 antibodies in my experimental system?

Rigorous validation of antibody specificity is essential for reliable research outcomes. Implement the following validation strategies:

Genetic approaches:

• IFRD1 knockdown/knockout controls: Use siRNA knockdown of IFRD1 as demonstrated in previous studies or CRISPR-Cas9 knockout cell lines to confirm signal reduction

• Overexpression systems: Compare signal in cells with normal versus overexpressed IFRD1 levels

• IFRD1 reporter systems: Use established reporter systems such as the X-gal staining in IFRD1 reporter mice for correlation with antibody signal

Biochemical validation:

• Peptide competition assays: Pre-incubate antibody with purified IFRD1 antigen or immunizing peptide to demonstrate signal reduction

• Multiple antibody comparison: Test multiple antibodies targeting different IFRD1 epitopes

• Western blot correlation: Confirm specificity by western blot showing expected 50-55 kDa band corresponding to IFRD1

Physiological validation:

• Induction experiments: Verify increased IFRD1 detection under conditions known to upregulate expression, such as:

  • RANKL stimulation in preosteoclasts

  • Glutamine starvation in hepatocellular carcinoma cells

• Tissue expression patterns: Confirm detection in tissues with known IFRD1 expression (e.g., superficial urothelial cell layer of the bladder)

Document validation results systematically using the following format:

What are the common pitfalls when working with FITC-conjugated antibodies for IFRD1 detection and how can they be addressed?

Several technical challenges can affect experiments with FITC-conjugated IFRD1 antibodies. Here are common issues and their solutions:

Photobleaching:

• Issue: FITC is susceptible to photobleaching during prolonged imaging

• Solution: Use anti-fade mounting media, minimize exposure times, consider alternative more photostable fluorophores for extended imaging sessions

Autofluorescence interference:

• Issue: Tissues may exhibit natural fluorescence in the FITC emission spectrum

• Solution: Implement tissue-specific autofluorescence reduction protocols such as Sudan Black B treatment or spectral unmixing during image acquisition

pH sensitivity:

• Issue: FITC fluorescence is pH-dependent, decreasing in acidic conditions

• Solution: Maintain consistent pH (ideally pH 8.0) in all buffers used for antibody incubation and washing steps

Fixation-induced epitope masking:

• Issue: IFRD1 epitopes may be masked during fixation

• Solution: Test multiple fixation protocols; consider antigen retrieval methods such as citrate buffer treatment for formalin-fixed tissues

Low signal-to-noise ratio:

• Issue: Weak specific signal relative to background

• Solution: Optimize antibody concentration through titration experiments, increase incubation time at 4°C, implement signal amplification systems

Cross-reactivity concerns:

• Issue: Potential cross-reactivity with related proteins

• Solution: Validate using IFRD1 knockout controls, perform careful bioinformatic analysis of epitope uniqueness

Subcellular localization accuracy:

• Issue: IFRD1 exhibits complex localization patterns dependent on cellular state

• Solution: Use subcellular markers to confirm localization, implement super-resolution microscopy techniques for detailed analysis

Based on insights from research using IFRD1 antibodies , key recommendations include careful optimization of fixation conditions (as demonstrated in bladder tissue preparations) and correlation with complementary approaches such as gene expression analysis to validate antibody-based findings.

How can FITC-conjugated IFRD1 antibodies be used to investigate its role in disease models?

IFRD1 has been implicated in several disease processes, making it an important research target. FITC-conjugated antibodies enable multiple investigative approaches:

Cancer research applications:
IFRD1 promotes tumor cell survival under glutamine starvation in hepatocellular carcinoma . Research approaches include:

• Treatment response monitoring: Assess changes in IFRD1 expression following glutaminase inhibitor (CB-839) treatment

• Metabolic stress pathway analysis: Correlate IFRD1 expression with markers of autophagy (given IFRD1's role in inhibiting autophagy by promoting proteasomal degradation of ATG14)

• Therapeutic target validation: Use FITC-conjugated antibodies to confirm IFRD1 knockdown efficacy in preclinical models

Viral infection studies:
IFRD1 is exploited by human papillomavirus for immune evasion . Research applications include:

• Viral immune evasion mechanism assessment: Track IFRD1 expression and localization during viral infection

• EGFR-IFRD1-NFκB pathway analysis: Monitor changes in IFRD1 expression following EGFR inhibitor (cetuximab) treatment

• Cytokine response correlation: Measure relationship between IFRD1 levels and pro-inflammatory cytokine production

Inflammatory disorder research:

• Immune cell activation studies: Monitor IFRD1 dynamics during immune cell activation

• Tissue-specific inflammation models: Investigate IFRD1 expression in models of bladder inflammation, given its role in bladder epithelial homeostasis

Methodological approach table:

What are the most effective strategies for multiplexing FITC-conjugated IFRD1 antibodies with other markers?

Multiplexing enables simultaneous detection of IFRD1 and other proteins to gain comprehensive insights into biological processes. Optimal strategies include:

Spectral compatibility planning:

• Complementary fluorophores: Pair FITC (Ex/Em: 495/519nm) with fluorophores having minimal spectral overlap:

  • Cy3 or PE (Ex/Em: ~550/570nm)

  • APC (Ex/Em: ~650/660nm)

  • Pacific Blue (Ex/Em: ~410/455nm)

• Sequential imaging: For confocal microscopy of challenging combinations, consider sequential acquisition to minimize bleed-through

Multi-parameter flow cytometry:
When investigating IFRD1 in immune contexts as described in HPV research :

• Combine FITC-IFRD1 with surface markers (CD45, CD3, etc.) using red/far-red fluorophores

• Add nuclear markers (transcription factors) with violet/UV-excited fluorophores

• Include functional readouts (cytokine production, phospho-proteins) with orange/red fluorophores

High-dimensional analysis approaches:

• Spectral unmixing: For confocal microscopy or spectral flow cytometry

• Iterative staining: Cyclic immunofluorescence protocols for high-parameter imaging

• Signal separation algorithms: Computational approaches to separate overlapping signals

Biologically relevant multiplexing panels:

Based on IFRD1 research findings, particularly valuable marker combinations include:

These multiplexing approaches allow researchers to simultaneously assess IFRD1 expression and its functional relationships with interacting proteins and pathways.

What statistical approaches are recommended for analyzing IFRD1 expression data from FITC-based detection methods?

For flow cytometry data:

• Population comparisons: Use non-parametric tests (Mann-Whitney U) for comparing median fluorescence intensity between experimental groups

• Multi-parameter analysis: Implement dimensionality reduction techniques (tSNE, UMAP) to identify cell populations with correlated IFRD1 expression patterns

• Correlation analysis: Spearman rank correlation for assessing relationships between IFRD1 and other markers

For immunofluorescence microscopy data:

• Image segmentation: Automated cell/nucleus identification followed by intensity measurements

• Spatial statistics: Nearest neighbor analysis for assessing clustering of IFRD1-positive cells

• Colocalization analysis: Pearson's correlation coefficient or Manders' overlap coefficient for quantifying IFRD1 colocalization with other proteins

For time-course experiments:

• Repeated measures ANOVA: For comparing IFRD1 expression changes over time between treatment groups

• Linear mixed models: To account for within-subject correlations in longitudinal studies

• Survival analysis: Kaplan-Meier with IFRD1 expression as stratification variable in disease progression studies

Sample size considerations:
Based on effect sizes observed in previous IFRD1 studies:

• Gene expression differences between WT and Ifrd1-/- mice showed 1.5-fold changes (FDR-adjusted p<0.05)

• For detecting similar effect sizes with 80% power at alpha=0.05, minimum sample sizes of 5-8 per group are typically required

Visualization recommendations:

• Box plots with individual data points: For comparing IFRD1 expression across experimental groups

• Heatmaps: For visualizing IFRD1 correlations with multiple other markers

• Violin plots: For displaying distribution characteristics of IFRD1 expression

How should researchers interpret changes in IFRD1 localization versus expression levels in experimental systems?

IFRD1 function is regulated by both expression levels and subcellular localization, requiring careful interpretation of experimental observations:

Distinguishing localization from expression changes:

Functional interpretation guidelines:

Context-specific interpretation:

• In HPV-infected cells: IFRD1 localization affects its ability to modulate RelA acetylation, with nuclear IFRD1 potentially indicating active NFκB pathway suppression

• In metabolically stressed cells: Cytoplasmic IFRD1 accumulation may indicate its role in regulating autophagy proteins like ATG14

• In urothelial cells: IFRD1 expression in superficial cell layers suggests role in terminal differentiation

Integrative analysis approach:

• Correlate localization patterns with functional readouts (e.g., target gene expression)

• Compare with known stimuli that affect IFRD1 localization (EGFR pathway activation, metabolic stress)

• Consider post-translational modifications that might affect localization (phosphorylation, acetylation)

Careful interpretation of both expression and localization data provides deeper insights into IFRD1's context-specific functions across different research models.