yipf5 Antibody

What is YIPF5 and what cellular functions does it regulate?

YIPF5 (also known as YIP1A, FINGER5, and SMAP-5) is a small five-span transmembrane domain protein that plays crucial roles in transport between the endoplasmic reticulum and Golgi apparatus. Recent studies have identified YIPF5 as a positive regulator of STING trafficking and essential for innate immune responses to DNA viruses . Additionally, YIPF5 forms complexes with other proteins including YIF1A and GOT1B to maintain Golgi structural integrity and facilitate protein transport through the secretory pathway .

What are the available types of YIPF5 antibodies and their molecular characteristics?

Currently available YIPF5 antibodies are primarily rabbit polyclonal antibodies that show reactivity with human, mouse, and rat samples. These antibodies are typically generated using immunogens derived from YIPF5 fusion proteins. The commercial antibodies recognize YIPF5 with an observed molecular weight of approximately 28-33 kDa, which aligns with the calculated molecular weight of 28 kDa for the 257 amino acid protein .

What applications are YIPF5 antibodies validated for in research?

YIPF5 antibodies have been validated for multiple experimental applications:

Note: Optimal dilutions are sample-dependent and should be determined empirically for each experimental system .

How should researchers optimize YIPF5 antibody protocols for investigating innate immune responses to DNA viruses?

When investigating YIPF5's role in innate immunity:

• Cell Selection: Use appropriate cell lines (THP-1, L929) or primary cells (BMDMs, MEFs) that express endogenous YIPF5 and STING.

• Stimulation Parameters: Stimulate cells with DNA viruses (e.g., HSV-1), synthetic dsDNA transfection, or cGAMP treatment based on experimental goals.

• Antibody Application:

  • For protein localization during trafficking: Optimize ICC/IF with paraformaldehyde fixation and Triton X-100 permeabilization

  • For detecting protein-protein interactions: Use co-immunoprecipitation with YIPF5 antibodies followed by Western blot analysis for potential binding partners (STING, components of COPII)

• Controls:

  • Include YIPF5 knockdown/knockout controls

  • Use RNA virus (e.g., SeV) stimulation as a negative control since YIPF5 specifically regulates DNA-triggered but not RNA-triggered innate immunity

For optimal results in co-localization studies, the timing of sample collection after stimulation is critical, as YIPF5-STING interaction is enhanced following DNA stimulation .

What strategies are effective for investigating YIPF5-STING interaction dynamics in cellular trafficking pathways?

Based on research findings, several effective strategies include:

• Sequential Immunoprecipitation: Use a two-step immunoprecipitation approach to isolate YIPF5-STING-COPII complexes.

  • First IP: Pull down YIPF5 complexes

  • Second IP: Use antibodies against interaction partners to confirm specificity

• Domain Mapping: Utilize the knowledge that C-terminal transmembrane domains of YIPF5 interact with the fourth transmembrane domain of STING to design truncation constructs for detailed interaction studies .

• Colocalization Analysis:

  • Perform time-course experiments after dsDNA stimulation to track YIPF5-STING colocalization

  • Use high-resolution microscopy (confocal, super-resolution) to visualize the spatial relationship between YIPF5, STING, and components of COPII at ER exit sites

• Trafficking Inhibition: Use Brefeldin A (BFA) to block ER-Golgi trafficking and compare with FLI-06 treatment to distinguish different stages of the secretory pathway where YIPF5 functions .

These approaches can reveal how YIPF5 facilitates STING recruitment to COPII-coated vesicles during DNA virus infection .

How can researchers address non-specific binding issues when using YIPF5 antibodies in Western blot applications?

To minimize non-specific binding:

• Optimization of Antibody Dilution: Test a concentration gradient (1:500-1:3000) to determine optimal signal-to-noise ratio for your specific sample type.

• Blocking Optimization:

  • Use 5% non-fat dry milk or BSA in TBST

  • For tissues with high endogenous biotin, add avidin-biotin blocking steps

• Membrane Washing: Implement stringent washing protocols with increased wash duration (5-10 minutes per wash, 3-5 times).

• Validation Through Knockdown: Include YIPF5 knockdown or knockout samples as negative controls to confirm the specificity of the detected bands at 28-33 kDa .

• Sample Preparation Considerations: YIPF5 is a membrane protein, so use appropriate lysis buffers containing mild detergents (e.g., NP-40 or Triton X-100) to maintain protein structure while ensuring sufficient extraction .

What are the critical considerations when interpreting YIPF5 immunohistochemistry results in different tissue types?

When interpreting IHC results:

• Antigen Retrieval Method Selection: Different tissues require different retrieval approaches:

  • For liver and brain tissues: TE buffer pH 9.0 is recommended

  • Alternative approach: Citrate buffer pH 6.0 for tissues with different fixation histories

• Expression Pattern Analysis:

  • YIPF5 typically shows perinuclear and vesicular staining patterns

  • Golgi localization is prominent in normal tissues

  • Altered localization patterns may occur in pathological conditions

• Tissue-Specific Considerations:

  • Brain tissue: Higher background due to lipid content; may require extended blocking

  • Liver tissue: High endogenous peroxidase activity requires thorough quenching

  • Cancer tissues: May show altered expression or localization patterns that reflect pathology rather than technical issues

• Multiple Antibody Validation: When possible, confirm findings with antibodies targeting different epitopes of YIPF5 to rule out epitope-specific artifacts.

How can YIPF5 antibodies be employed to investigate its role in diabetes and microcephaly models?

Recent research has linked YIPF5 mutations to neonatal diabetes and microcephaly. When investigating these connections:

• Patient-Derived Models:

  • Use YIPF5 antibodies in iPSC-derived β-cells from patients with YIPF5 mutations

  • Compare subcellular localization and trafficking patterns with wild-type controls

• Mechanistic Studies:

  • Immunofluorescence combined with proinsulin staining to assess proinsulin trafficking defects

  • Co-localization with ER stress markers (BiP/GRP78, CHOP) to evaluate ER stress mechanisms

• Animal Models:

  • Apply antibodies to tissue sections from YIPF5 mutant models (e.g., p.W218R mutation in rabbits)

  • Quantitative analysis of brain development and β-cell mass/function correlations

• Therapeutic Intervention Assessment:

  • Monitor changes in YIPF5 localization and proinsulin trafficking in response to ER stress modulators

  • Evaluate whether insulin sensitizers can alleviate ER stress by reducing insulin production demand

What methods are effective for investigating YIPF5-GOT1A/B complex formation in ER-Golgi trafficking studies?

To investigate the recently identified YIPF5-GOT1A/B complex:

• Complex Isolation Strategies:

  • Sequential immunoprecipitation with antibodies against YIPF5 followed by GOT1A or GOT1B

  • Blue native PAGE to preserve native complex structures

  • Size exclusion chromatography to isolate intact complexes

• Drug Perturbation Analysis:

  • Treat cells with FLI-06 to disrupt the complex and analyze changes in composition

  • Compare the effects of different trafficking inhibitors on complex stability

• Mutational Analysis:

  • Generate constructs with mutations in key interaction domains

  • Use YIPF5 antibodies to assess how mutations affect complex formation and trafficking function

• Dynamics Assessment:

  • Pulse-chase experiments with fluorescently labeled cargo proteins

  • Live-cell imaging with tagged YIPF5 and GOT1A/B to track complex movement

This complex appears to play a role in a transcription-independent mechanism of ER export, and understanding its composition and regulation could provide insights into fundamental cell biology mechanisms .

How can YIPF5 antibodies be utilized to explore its potential roles beyond vesicular trafficking?

YIPF5 research is expanding beyond its established roles:

• Antiviral Immunity Assessment:

  • Use YIPF5 antibodies to track its redistribution during viral infection

  • Investigate potential roles in responses to different DNA virus families (herpesviruses, adenoviruses)

  • Explore whether YIPF5 is targeted by viral evasion mechanisms

• Cell Stress Response Studies:

  • Examine YIPF5 localization during different cellular stresses (ER stress, oxidative stress)

  • Investigate potential stress-induced post-translational modifications using phospho-specific or other modification-specific antibodies

• Developmental Biology Applications:

  • Track YIPF5 expression patterns during embryonic development with particular focus on brain and pancreas

  • Correlate expression with developmental milestones in these tissues

• Cancer Research:

  • Analyze YIPF5 expression and localization in tumor vs. normal tissues

  • Investigate whether altered YIPF5 function contributes to cancer progression through disruption of secretory pathway functions

What are the most promising methodological advances for studying the dynamic interactome of YIPF5 in living cells?

Emerging methodologies for studying YIPF5's dynamic interactions include:

• Proximity Labeling Approaches:

  • BioID or TurboID fusion with YIPF5 to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal analysis of interaction partners

• Advanced Imaging Techniques:

  • FRET/FLIM analysis of YIPF5 interactions with STING, COPII components, or GOT1A/B

  • Lattice light-sheet microscopy for high-speed, low-phototoxicity imaging of trafficking dynamics

  • Super-resolution microscopy techniques (STORM, PALM) to visualize nanoscale organization

• Optogenetic Control Systems:

  • Light-inducible protein interaction systems to trigger or disrupt YIPF5 complex formation

  • Optogenetic control of YIPF5 localization to study spatial requirements of function

• Single-Molecule Tracking:

  • Use antibody fragments conjugated to quantum dots for long-term tracking of individual YIPF5 molecules

  • Correlate movement patterns with cellular responses to stimuli

These approaches can provide unprecedented insights into how YIPF5 dynamically regulates trafficking processes and immune responses in real-time.