IZUMO4 Antibody

What is IZUMO4 and how does it relate to other IZUMO family proteins?

IZUMO4 is a member of the IZUMO protein family, which includes four paralogues (IZUMO1-4). Unlike IZUMO1, 2, and 3, which are transmembrane proteins primarily expressed in the testis, IZUMO4 is a soluble protein expressed in the testis and other tissues . IZUMO4 has an N-terminal domain with significant homology to the N-terminal domain of IZUMO1, which is known to be essential for sperm-egg fusion during fertilization . While IZUMO1 has been extensively characterized as crucial for fertilization, IZUMO4's precise physiological role is still being investigated.

What applications are IZUMO4 antibodies validated for?

IZUMO4 antibodies have been validated for multiple applications, including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

Most commercially available IZUMO4 antibodies have been tested with human samples, and some also show reactivity with rat tissues .

What is the typical molecular weight of IZUMO4 on Western blots?

IZUMO4 typically appears in two isoforms on Western blots: approximately 50 kDa and 56 kDa . This pattern is distinct from other IZUMO family members. For example, IZUMO1 typically shows bands at approximately 56 kDa (non-phosphorylated) and 60 kDa (phosphorylated), while IZUMO3 shows bands at approximately 55 kDa and 37 kDa .

How should I optimize antibody dilutions for different IZUMO4 detection techniques?

Optimal dilution ranges vary by application:

• ELISA: 1:2000-1:10000

• Western Blot: 1:500-1:5000

• Immunohistochemistry: 1:20-1:200

• Immunofluorescence: 1:50-1:200

These ranges should be used as starting points for optimization. When establishing optimal conditions, perform a dilution series to determine the concentration that provides the highest signal-to-noise ratio for your specific sample type. For Western blots, verify band specificity by including positive controls (e.g., testis tissue or recombinant IZUMO4) and ensuring bands appear at the expected molecular weights (approximately 50 kDa and 56 kDa) .

What is the most effective immunogen for generating high-specificity IZUMO4 antibodies?

Most effective IZUMO4 antibodies are generated using recombinant human IZUMO4 protein (amino acids 16-214) as the immunogen . This region corresponds to the extracellular domain and contains the IZUMO domain, which is crucial for protein-protein interactions. For polyclonal antibodies, purification using protein G affinity chromatography yields antibodies with >95% purity, providing high specificity for IZUMO4 detection with minimal cross-reactivity to other IZUMO family members .

How can I distinguish between IZUMO4 and other IZUMO family members in my experiments?

To ensure specificity:

• Select antibodies raised against unique regions of IZUMO4 not conserved among other family members

• Validate antibody specificity using recombinant proteins of all IZUMO family members

• Perform Western blot analysis checking for distinct patterns:

  • IZUMO1: ~56 kDa and ~60 kDa bands

  • IZUMO3: ~55 kDa and ~37 kDa bands

  • IZUMO4: ~50 kDa and ~56 kDa bands

• Include appropriate controls in immunofluorescence studies, noting that IZUMO4 localization differs from IZUMO1 (anterior acrosome and equatorial region) and IZUMO3 (post-acrosomal region)

How does the acrosome reaction affect IZUMO4 expression and localization on sperm?

Research has shown that both isoforms of IZUMO4 are reduced in acrosome-reacted sperm, with the larger isoform (~56 kDa) being almost completely absent . This pattern differs significantly from IZUMO1, which undergoes redistribution but remains present after the acrosome reaction, and from IZUMO3, which is largely retained on acrosome-reacted sperm . These findings suggest IZUMO4 may be an intra-acrosomal protein released during the acrosome reaction, making it a potential marker for studying this critical fertilization step. When designing experiments to study IZUMO4 during fertilization, researchers should time their analysis carefully to capture protein dynamics before and after the acrosome reaction.

What are the optimal sample preparation methods for studying IZUMO4 in sperm?

Due to IZUMO4's apparent location within the acrosome and its loss during sonication, researchers should consider:

• For intact IZUMO4 analysis:

  • Use gentle fixation methods (2-4% paraformaldehyde)

  • Avoid harsh detergents or sonication that may disrupt the acrosome

  • Process samples quickly to prevent spontaneous acrosome reactions

• For subcellular localization studies:

  • Compare fractionated sperm heads and tails using differential centrifugation and velocity sedimentation

  • Include acrosomal markers (e.g., acrosin) as controls

  • Use permeabilization agents selectively to distinguish intra-acrosomal from membrane-associated proteins

How can IZUMO4 antibodies be applied to study protein complex formation?

IZUMO4 forms protein complexes with IZUMO3 and potentially other sperm proteins . To study these complexes:

• Use native gel electrophoresis under mildly denaturing conditions to preserve protein-protein interactions

• Perform co-immunoprecipitation with IZUMO4 antibodies followed by mass spectrometry to identify interaction partners

• Apply chemical crosslinking before immunoprecipitation to stabilize transient interactions

• Use proximity ligation assays (PLA) for in situ detection of IZUMO4-partner interactions

• Consider bimolecular fluorescence complementation (BiFC) for studying IZUMO4 complex formation in live cells

What explains discrepancies in IZUMO4 detection between different sample types?

Researchers may observe variations in IZUMO4 detection due to:

• Tissue-specific post-translational modifications: Unlike other IZUMO family members, IZUMO4 is expressed in multiple tissues and may undergo different modifications affecting antibody recognition

• Protein complex formation: As IZUMO4 forms complexes, epitope accessibility may be affected

• Sample preparation methods: IZUMO4 can be lost during processing, particularly with methods that disrupt the acrosome

• Antibody specificity: Some antibodies may detect only specific isoforms or conformations

If inconsistent results are observed across tissues, verify antibody specificity using recombinant IZUMO4 protein as a positive control and compare results using antibodies targeting different epitopes .

How should negative or inconclusive results with IZUMO4 antibodies be interpreted?

When encountering negative results:

• Verify antibody activity using positive controls (testis lysate for endogenous protein or recombinant IZUMO4)

• Consider tissue-specific expression levels (IZUMO4 is expressed in testis but also in other tissues at varying levels)

• Assess sample preparation methods (harsh methods may cause IZUMO4 loss)

• Test different fixation and permeabilization methods for immunofluorescence studies

• Evaluate whether acrosome reaction status affects detection (IZUMO4 levels decrease after acrosome reaction)

What are the key controls necessary for validating IZUMO4 antibody specificity?

For rigorous validation:

These controls are essential for publication-quality research and help distinguish true signals from artifacts .

How conserved is IZUMO4 across species and what implications does this have for antibody selection?

IZUMO4 shows species variation that researchers must consider when selecting antibodies. According to commercial antibody specifications, many IZUMO4 antibodies demonstrate reactivity to human samples, with some also cross-reacting with rat samples . When working with other species, researchers should carefully evaluate sequence homology in the epitope region and validate antibody cross-reactivity empirically.

For comparative studies across species, consider:

• Selecting antibodies raised against conserved regions

• Testing specificity in each species of interest

• Using multiple antibodies targeting different epitopes to confirm findings

• Complementing antibody studies with genetic approaches when possible

How do the structures and functions of IZUMO family proteins compare?

The IZUMO family contains four paralogues with varying structures and functions:

Understanding these differences is crucial when designing experiments targeting specific IZUMO family members and interpreting results in the context of reproductive biology .

How can IZUMO4 antibodies contribute to understanding infertility mechanisms?

IZUMO4 antibodies can illuminate several aspects of fertility research:

• Expression analysis in infertile males versus fertile controls to identify potential correlations between IZUMO4 expression patterns and fertility outcomes

• Investigating IZUMO4's potential interactions with JUNO, the oocyte receptor for IZUMO1, or other egg surface proteins

• Examining changes in IZUMO4 during sperm capacitation and acrosome reaction across various fertility disorders

• Assessing IZUMO4 as a potential biomarker for specific types of male infertility

• Studying potential roles of IZUMO4 antibodies in immune-related infertility

This research could provide insights into unexplained infertility cases and potentially inform new diagnostic or therapeutic approaches.

What is known about the interaction between IZUMO4 and other proteins during fertilization?

While IZUMO1's interaction with JUNO on the egg surface is well established , IZUMO4's interactions during fertilization remain less clear. Research indicates:

• IZUMO4 forms complexes with IZUMO3 on sperm

• The IZUMO domain of IZUMO4 can form dimers, suggesting potential homotypic interactions

• As a soluble intra-acrosomal protein, IZUMO4 may be released during the acrosome reaction to interact with extracellular proteins

• Unlike IZUMO1, IZUMO4 does not appear to interact with JUNO according to systematic interaction screens

To further investigate these interactions, researchers could employ techniques such as proximity labeling, co-immunoprecipitation with IZUMO4 antibodies followed by mass spectrometry, or yeast two-hybrid screening to identify novel interaction partners.