wee2 Antibody

What is WEE2 and why is it important in reproductive research?

WEE2, also known as WEE1B, is an oocyte-specific protein tyrosine kinase belonging to the protein kinase superfamily within the Ser/Thr protein kinase family and WEE1 subfamily. It functions as a key regulator of meiosis during both prophase I and metaphase II by phosphorylating and inhibiting CDK1/CDC2. WEE2 plays a dual regulatory role in oocyte maturation: it maintains immature oocytes in prophase I arrest before the luteinizing hormone surge and facilitates exit from metaphase II arrest during fertilization . This critical function in meiotic regulation makes WEE2 particularly important in reproductive biology research, as it represents a highly specific target for potential non-hormonal contraceptive development . Unlike its somatic counterpart WEE1, WEE2 expression is restricted to oocytes, making it an excellent candidate for targeted interventions in female reproduction without systemic effects.

What are the structural and functional differences between WEE2 and WEE1?

WEE2 and WEE1 share structural similarities as both belong to the WEE1 subfamily of protein kinases, but they have distinct expression patterns and functional roles. WEE1 is widely expressed in somatic cells and regulates mitotic cell cycles, while WEE2 is oocyte-specific and regulates meiotic progression . While both kinases phosphorylate CDK1 at Tyr-15 to inhibit its activity, they do so in different cellular contexts - WEE1 in mitotic cells and WEE2 specifically in meiotic oocytes . This differential expression pattern allows for the development of selective inhibitors that can target WEE2 without affecting WEE1, which would minimize off-target effects in somatic cells. Researchers have used homology modeling based on WEE1's crystal structure to predict WEE2's binding properties, showing that despite similarities, the ATP-binding sites of these kinases have sufficient differences to develop selective inhibitors .

How do I select the appropriate WEE2 antibody for my specific application?

Selecting the appropriate WEE2 antibody depends on your experimental application, species of interest, and detection methods. For Western blot applications, the 55119-1-AP WEE2 antibody has been validated for human samples at dilutions of 1:500-1:1000 . This rabbit polyclonal antibody recognizes the full-length WEE1 homolog 2 protein and detects a band at approximately 63-66 kDa . When selecting an antibody, verify:

• Reactivity with your species of interest (the 55119-1-AP is validated for human samples)

• Application compatibility (WB, ELISA, etc.)

• Specificity (confirm it doesn't cross-react with WEE1)

• Validation data in relevant tissues (the 55119-1-AP has been tested in Jurkat cells and human ovary tissue)

For validation of antibody specificity, consider using positive controls such as human ovary tissue, which naturally expresses WEE2, and negative controls such as tissues that don't express WEE2 . It's also recommended to titrate the antibody in your specific experimental system to determine optimal concentration.

What are the optimal storage and handling conditions for WEE2 antibodies?

To maintain optimal activity of WEE2 antibodies, proper storage and handling are essential. For the 55119-1-AP WEE2 antibody, the recommended storage conditions are:

• Store at -20°C in the provided storage buffer (0.1M NaHCO3, 0.1M glycine, 0.02% sodium azide, and 50% glycerol at pH 7.3)

• Aliquoting is unnecessary for -20°C storage for this particular formulation

• Note that some preparations (20μl sizes) contain 0.1% BSA as a stabilizer

When handling antibodies, follow these best practices:

• Avoid repeated freeze-thaw cycles

• Maintain sterile conditions when pipetting

• Allow the antibody to reach room temperature before opening the vial

• Briefly centrifuge to collect liquid at the bottom of the tube

• Return to storage promptly after use

• Check expiration dates and monitor for signs of degradation (cloudiness, precipitation)

For long-term projects, consider requesting larger volumes and making working aliquots to extend shelf life and maintain consistency across experiments.

What are the validated experimental applications for WEE2 antibodies?

Current validated applications for the 55119-1-AP WEE2 antibody include Western Blot (WB) and ELISA applications . In Western blot applications, this antibody detects WEE2 protein at its expected molecular weight of 63-66 kDa in human samples, particularly in ovary tissue and Jurkat cells . While immunohistochemistry applications aren't explicitly listed in the product information, the antibody's specificity for WEE2 suggests it might be suitable for this application after proper validation.

For researchers studying WEE2 kinase activity rather than just protein expression, enzyme-linked immunosorbent assays have been developed to measure WEE2 phosphorylation activity on cyclin-dependent kinase 1 (CDK1/CDC2) . These functional assays detect only the phosphorylated form of tyrosine 15 of CDK1 using anti-phospho-tyrosine monoclonal antibodies, allowing quantitative measurement of WEE2 kinase activity . These assays are particularly valuable when evaluating potential WEE2 inhibitors, as they provide direct measurement of functional impact rather than simply detecting protein presence.

How can I optimize Western blot protocols for WEE2 detection?

To optimize Western blot protocols for WEE2 detection using the 55119-1-AP antibody, consider the following methodology:

• Sample preparation:

  • Use fresh tissue/cells when possible

  • For ovary tissue samples, ensure proper homogenization in RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if examining phosphorylation status

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels to properly resolve the 63-66 kDa WEE2 protein

  • Load appropriate positive controls (human ovary tissue) and negative controls

• Transfer and blocking:

  • Use PVDF membranes for optimal protein binding

  • Block with 5% non-fat milk or BSA in TBST for at least 1 hour at room temperature

• Primary antibody incubation:

  • Dilute the 55119-1-AP antibody at 1:500-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Consider titrating the antibody concentration to determine optimal signal-to-noise ratio

• Detection:

  • Use appropriate HRP-conjugated secondary antibodies (anti-rabbit IgG)

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Expected molecular weight: 63-66 kDa

For troubleshooting, if background is high, try increasing blocking time or washing steps. If signal is weak, consider longer exposure times, increased antibody concentration, or enhanced detection systems.

How can I validate the specificity of WEE2 antibody staining?

Validating the specificity of WEE2 antibody staining is crucial to ensure reliable experimental results. Consider these approaches:

• Positive and negative tissue controls:

  • Positive control: Human ovary tissue (known to express WEE2)

  • Negative control: Tissues not expected to express WEE2 (as it's oocyte-specific)

• Knockdown/knockout validation:

  • Use RNA interference to knock down WEE2 expression

  • Verify reduced antibody signal in knockdown samples compared to controls

  • This approach has been used successfully in rhesus macaque studies where WEE2 downregulation via dsRNA-mediated RNAi led to meiotic resumption in GV oocytes

• Blocking peptide competition:

  • Pre-incubate the antibody with excess immunogenic peptide

  • This should significantly reduce or eliminate specific staining

• Correlation with mRNA expression:

  • Compare antibody staining with WEE2 mRNA levels using qPCR

  • Tissues with higher mRNA expression should show stronger antibody signal

• Multiple antibody validation:

  • Use a second antibody targeting a different epitope of WEE2

  • Similar staining patterns increase confidence in specificity

These validation approaches should be documented and included in publications to support the reliability of your findings.

How can WEE2 antibodies be used to investigate meiotic regulation in oocytes?

WEE2 antibodies are valuable tools for investigating meiotic regulation in oocytes at multiple levels:

• Expression pattern analysis:

  • Use WEE2 antibodies in immunofluorescence to track spatiotemporal expression during oocyte maturation

  • Studies in rhesus macaques have characterized WEE2 expression patterns throughout oocyte development

• Functional studies combining antibody detection with genetic manipulation:

  • RNA interference approaches have shown that WEE2 downregulation leads to meiotic resumption in germinal vesicle (GV) oocytes even in the presence of phosphodiesterase 3 inhibitors

  • Conversely, WEE2 overexpression delays reentry of oocytes into meiosis in both mice and monkeys

• Phosphorylation state analysis:

  • Use WEE2 antibodies alongside phospho-CDK1 (Tyr15) antibodies to correlate WEE2 levels with its kinase activity

  • This approach helps establish the causative relationship between WEE2 expression and CDK1 inhibition

• Protein-protein interaction studies:

  • Immunoprecipitation with WEE2 antibodies can identify interaction partners regulating meiotic progression

  • Co-immunoprecipitation followed by mass spectrometry can reveal novel regulatory complexes

These approaches have established WEE2 as a conserved oocyte-specific meiosis inhibitor functioning downstream of cAMP in primates , providing insight into fundamental mechanisms of female fertility regulation.

What role can WEE2 antibodies play in contraceptive development research?

WEE2 antibodies are instrumental in contraceptive development research through several key applications:

• Target validation studies:

  • WEE2 antibodies help confirm the oocyte-specific expression of WEE2, supporting its potential as a selective contraceptive target

  • Immunohistochemistry using these antibodies demonstrates minimal expression in non-reproductive tissues, suggesting limited off-target effects

• Inhibitor screening workflows:

  • Antibody-based ELISAs measuring WEE2 phosphorylation activity on CDK1 are crucial for evaluating candidate WEE2 inhibitors

  • These assays provide quantitative measurement of inhibitor potency and selectivity over WEE1

• Mechanism of action studies:

  • WEE2 antibodies help elucidate how inhibitors affect metaphase II exit during fertilization

  • In vitro fertilization studies using bovine ova complement antibody-based assays to determine inhibition of metaphase II exit

• Selectivity assessment:

  • Comparing inhibitor effects on WEE2 versus WEE1 using parallel antibody-based assays helps identify compounds with optimal selectivity profiles

  • Cell-proliferation assays detect off-target effects against WEE1 in somatic (mitotic) cells

This research has identified promising candidates like compound 2 (PD-166285) and its analogs (compounds 12 and 16) as potential non-hormonal contraceptives targeting WEE2 .

How can I distinguish between WEE2 and WEE1 activity in complex biological samples?

Distinguishing between WEE2 and WEE1 activity in complex biological samples requires careful experimental design:

• Differential expression analysis:

  • WEE2 is oocyte-specific while WEE1 is expressed in somatic cells

  • In mixed samples, cell sorting or microdissection before antibody-based detection can help separate populations

• Selective inhibition approach:

  • Use WEE1-specific inhibitors (like MK-1775) as controls to differentiate activity

  • Researchers have used differential scanning fluorimetry (DSF) to assess compound binding specificity between WEE2 and WEE1 kinase domains

• Enzyme-linked immunosorbent assays (ELISAs):

  • Parallel ELISAs using recombinant WEE2 (5 μg) and human WEE1 protein (40 mUnits) with the same inhibitor concentration (1 μM) allows comparison of inhibitory effects

  • Detection of phosphorylated CDK1 (Tyr15) with anti-phospho-tyrosine monoclonal antibodies provides quantitative measurement of kinase activity

• Functional validation in different cell types:

  • WEE2 inhibition affects meiosis in oocytes

  • WEE1 inhibition affects mitosis in somatic cells

  • This biological distinction allows validation through cell-type specific functional assays

These approaches helped researchers identify compounds with preferential binding to WEE2 over WEE1, demonstrating the feasibility of developing selective inhibitors despite the structural similarities between these kinases .

What are the best approaches for studying WEE2 mutations identified in clinical cases?

Several WEE2 mutations have been identified in clinical cases of fertilization failure, providing valuable research opportunities. Optimal approaches for studying these mutations include:

• Recombinant protein expression systems:

  • Express wild-type and mutant WEE2 proteins using baculovirus or mammalian expression systems

  • Use WEE2 antibodies to confirm expression and purify proteins for functional assays

  • This approach allows direct comparison of kinase activity between wild-type and mutant proteins

• Structural analysis:

  • Apply homology modeling techniques similar to those used in inhibitor development

  • Compare predicted structural changes in mutants, particularly mutations at p.His337 (a common frameshift mutation site) and p.Arg410Trp (associated with "leaky" blockade of metaphase II exit)

  • Use differential scanning fluorimetry to assess protein stability changes in mutants

• CRISPR/Cas9 genome editing:

  • Introduce clinical mutations into animal models or cell lines

  • Use WEE2 antibodies to confirm expression levels of mutant proteins

  • Evaluate functional consequences in oocyte maturation and fertilization

• Patient sample analysis:

  • When available, analyze oocytes from patients with WEE2 mutations

  • Immunofluorescence with WEE2 antibodies can detect abnormal localization or expression

  • Combined with CDK1 phosphorylation status analysis to assess functional impact

These approaches can provide insights into how mutations impact WEE2 structure, stability, and function, potentially informing both reproductive medicine and contraceptive development.

What are common issues in WEE2 antibody applications and how can they be resolved?

Researchers working with WEE2 antibodies may encounter several common challenges:

• Low signal intensity:

  • Increase antibody concentration (try 1:500 instead of 1:1000 for Western blot)

  • Extend incubation time (overnight at 4°C)

  • Use enhanced detection systems (high-sensitivity ECL substrates)

  • Ensure sample contains sufficient WEE2 (human ovary tissue as positive control)

• Non-specific binding:

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Increase washing steps (5 × 5 minutes with TBST)

  • Perform antibody titration to find optimal concentration

  • Pre-absorb antibody with non-specific proteins

• Inconsistent results:

  • Standardize protein extraction protocols

  • Use fresh samples when possible

  • Maintain consistent gel loading amounts

  • Include loading controls and positive controls in every experiment

• Cross-reactivity with WEE1:

  • Validate specificity using WEE1-expressing and WEE2-expressing tissues

  • Consider using monoclonal antibodies with confirmed specificity

  • Perform parallel detection with both WEE1 and WEE2 antibodies to confirm distinct patterns

• Difficulty detecting phosphorylated substrates:

  • Include phosphatase inhibitors in all buffers

  • Use phospho-specific antibodies (e.g., for phospho-CDK1) alongside WEE2 antibodies

  • Consider using functional assays like the ELISA-based systems described for inhibitor screening

Careful optimization of these parameters will improve reproducibility and reliability of WEE2 antibody applications.

How should I evaluate contradictory data when using WEE2 antibodies?

When faced with contradictory data using WEE2 antibodies, follow this systematic approach:

• Antibody validation assessment:

  • Confirm antibody specificity using multiple approaches (western blot, immunoprecipitation)

  • Verify batch-to-batch consistency by requesting validation data from manufacturers

  • Consider using alternative antibodies targeting different epitopes of WEE2

• Technical considerations:

  • Standardize sample preparation protocols across experiments

  • Document exact conditions (buffers, incubation times, detection methods)

  • Run side-by-side comparisons of contradictory conditions

• Biological variability analysis:

  • Consider developmental stage differences (WEE2 expression changes during oocyte maturation)

  • Evaluate species differences (studies in mice, monkeys, and humans might show variations)

  • Assess sample heterogeneity (mixed cell populations might obscure results)

• Functional validation approach:

  • Complement antibody-based detection with functional assays

  • Correlate WEE2 protein levels with CDK1 phosphorylation status

  • Validate with genetic approaches (RNAi, CRISPR) targeting WEE2

• Collaborative investigation:

  • Consult with other laboratories using the same antibodies

  • Share protocols and troubleshooting experiences

  • Consider independent validation of critical findings

This systematic approach helps distinguish between technical artifacts and true biological phenomena when investigating contradictory results.

What are the most reliable quantification methods for WEE2 expression studies?

For reliable quantification of WEE2 expression, researchers should consider these methodological approaches:

• Western blot quantification:

  • Use infrared fluorescence-based detection systems (e.g., LI-COR) for wider linear dynamic range

  • Include standard curves of recombinant WEE2 protein at known concentrations

  • Normalize to appropriate loading controls (GAPDH, β-actin)

  • Use software like ImageJ for densitometric analysis with background subtraction

• ELISA-based quantification:

  • Develop sandwich ELISAs using validated WEE2 antibodies

  • Include standard curves with recombinant WEE2

  • Ensure sample matrix matching between standards and unknowns

  • This approach offers higher throughput than Western blotting

• Mass spectrometry-based approaches:

  • Use targeted proteomics (selected reaction monitoring) for absolute quantification

  • Include isotopically labeled peptide standards corresponding to unique WEE2 regions

  • This approach offers high specificity and multiplexing capability

• qPCR correlation:

  • While measuring mRNA rather than protein, qPCR can complement antibody-based methods

  • Correlate protein levels with mRNA expression for more complete expression profile

  • Use multiple reference genes for normalization

• Quantitative immunofluorescence:

  • Use confocal microscopy with standardized acquisition parameters

  • Include calibration standards in each imaging session

  • Apply automated image analysis algorithms for unbiased quantification

Each method has strengths and limitations; combining multiple approaches provides the most reliable quantification of WEE2 expression.

How might WEE2 antibodies contribute to understanding age-related fertility decline?

WEE2 antibodies can significantly contribute to understanding age-related fertility decline through several research approaches:

• Comparative expression studies:

  • Use WEE2 antibodies to quantify expression levels in oocytes from young versus aged animals/humans

  • Immunofluorescence can reveal changes in subcellular localization that might correlate with reduced function

  • Western blot analysis can detect potential age-related post-translational modifications

• Functional correlation studies:

  • Combine WEE2 antibody detection with phospho-CDK1 antibodies to assess kinase activity

  • Determine if age-related fertility decline correlates with altered WEE2-mediated regulation

  • This approach can establish whether WEE2 dysfunction contributes to age-related meiotic errors

• Interactome analysis:

  • Use WEE2 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Compare protein interaction networks between young and aged oocytes

  • Identify age-related changes in regulatory complexes that might affect WEE2 function

• Intervention studies:

  • After identifying age-related changes, use WEE2 antibodies to monitor responses to interventions

  • This could include pharmacological approaches or genetic modifications designed to restore normal WEE2 function

These research directions may uncover whether altered WEE2 function contributes to the increased aneuploidy and reduced developmental competence observed in oocytes from older females, potentially leading to new fertility preservation strategies.

What are the prospects for developing WEE2-targeted therapies beyond contraception?

While current research focuses on WEE2 inhibitors as potential non-hormonal contraceptives , WEE2 antibodies can facilitate exploration of additional therapeutic applications:

• Fertility enhancement strategies:

  • For women with certain types of fertilization failure, modulating WEE2 activity might improve outcomes

  • WEE2 antibodies can help screen for compounds that modify rather than completely inhibit WEE2 function

  • Patient-specific diagnostics could identify individuals who might benefit from WEE2-targeted therapies

• In vitro maturation optimization:

  • WEE2 antibodies can monitor protein levels and localization during in vitro oocyte maturation

  • This could lead to culture condition improvements that better support normal WEE2 function

  • Particularly relevant for fertility preservation in cancer patients

• Diagnostic applications:

  • Development of diagnostic tests based on WEE2 function or expression

  • WEE2 antibodies could be used in immunoassays to detect abnormalities in oocytes

  • This might help identify specific causes of unexplained infertility

• Combination therapies:

  • WEE2 antibodies can help evaluate synergistic effects of targeting multiple meiotic regulators

  • This could lead to more effective interventions for various reproductive disorders

The table below summarizes the current WEE2 inhibitor candidates identified through screening, highlighting their potential for development beyond contraception: