PCSK4 Antibody

What is the molecular weight of PCSK4 protein targeted by PCSK4 antibodies?

The molecular weight of PCSK4 varies depending on its processing state. The pro-PCSK4 has a calculated molecular weight of approximately 72-83 kDa, while the mature PCSK4 has an observed molecular weight of 54 kDa as confirmed by Western blot analysis . This 54 kDa form is the functionally active enzyme found in the acrosomal membrane of spermatozoa. The difference between calculated and observed molecular weights is due to post-translational modifications and proteolytic processing that occurs during PCSK4 maturation . When designing experiments, researchers should be aware of this variation to properly identify the protein in their assays.

Which applications are most reliable for PCSK4 antibody detection in reproductive tissue samples?

Based on available research data, the following applications show high reliability for PCSK4 detection in reproductive tissues:

For consistent results in human sperm samples, immunohistochemistry techniques using DAB immunostaining have successfully confirmed PCSK4 expression on the sperm acrosome membrane . When working with rodent tissues, WB appears to be the most validated application with commercially available antibodies .

How should researchers design controls when using PCSK4 antibodies in fertility research?

When designing fertility research experiments involving PCSK4 antibodies, include these essential controls:

• Positive tissue controls: Human testis tissue or human placenta tissue should be used as positive controls as they reliably express PCSK4 .

• Negative controls:

  • Primary antibody omission

  • Tissues known not to express PCSK4

  • Preabsorption with immunizing peptide where available

• Specificity validation: Western blot confirmation of the 54 kDa band should precede functional studies to ensure antibody specificity .

• Functional controls: For fertilization studies, compare PCSK4 antibody-treated sperm with untreated controls in oocyte binding assays, as demonstrated in research showing that "spermatozoa given PCSK4 antibodies moved away from the oocyte extract compared with the controls whose spermatozoa were not treated" .

What are the optimal immunohistochemistry conditions for detecting PCSK4 in sperm samples?

Based on published protocols, the following conditions provide optimal PCSK4 detection in sperm samples:

• Sample preparation:

  • Fix samples in PBS-buffered fixatives

  • For enhanced antigen retrieval, use TE buffer pH 9.0 or citrate buffer pH 6.0

• Blocking conditions:

  • 1% BSA or 1% NGS in PBS for 10-30 minutes at room temperature

  • Wash in PBS pH 7.4 (3 × 5 minutes)

• Primary antibody incubation:

  • Anti-PCSK4 antibody at 1:50-1:500 dilution

  • Incubate for 1 hour at room temperature

• Visualization:

  • DAB immunostaining for bright-field microscopy

  • For fluorescence studies, rhodamine-labeled secondary antibodies have been successfully used for laser scanning microscopy

• Counterstaining:

  • Nuclear counterstaining improves visualization of PCSK4 localization relative to sperm head

How can researchers use PCSK4 antibodies to investigate the mechanism of sperm-egg interaction?

To investigate sperm-egg interaction mechanisms using PCSK4 antibodies:

• Receptor binding assays: Use purified PCSK4 antibodies to block the PCSK4 enzyme on sperm acrosomal membranes. This blocking "can inhibit the binding of PCSK4 to its receptors in the initiation process of the acrosome reaction when the spermatozoa approach the oocyte by penetrating the pellucida zone of the ZP3 receptor" .

• Fluorescence localization studies: Use rhodamine-labeled antibodies to visualize the binding pattern on the "membrane area of the spermatozoa head" during the fertilization process .

• Proteolytic activity assays: Measure PCSK4 enzyme activity using fluorogenic substrates like "Boc-RVRR-MCA" in the presence and absence of blocking antibodies to determine inhibition kinetics .

• Acrosome reaction analysis: Assess how anti-PCSK4 antibodies affect "acrosome reaction disturbance" through morphological and biochemical assays, as the "bond between PCSK4 and its antibodies causes a conformational change in the spermatozoa cell membrane protein" .

• In vitro fertilization models: Compare fertilization rates between antibody-treated and control sperm to quantify the impact of PCSK4 inhibition on fertility outcomes.

What methodological approaches resolve contradictions in PCSK4 expression data across different tissue types?

Researchers have encountered contradictory results regarding PCSK4 expression in non-reproductive tissues. To resolve these contradictions:

• Multi-technique validation: Combine different detection methods:

  • Western blot for protein size confirmation

  • qRT-PCR for mRNA quantification

  • Immunohistochemistry for spatial localization

  • Mass spectrometry for definitive protein identification

• Isoform-specific detection: Design experiments to differentiate between potential PCSK4 isoforms, similar to studies of "PACE4-altCT" that showed contradictory expression in malignant thyroid nodules .

• Species-specific considerations: Account for interspecies differences, as demonstrated with PCSK1/PC1/3, where "the presence of Arg vs Gln at the P2 residue renders human, but not mouse proGHRH susceptible to furin-mediated cleavage" .

• Standardized sample collection: Use consistent processing protocols to minimize technical variability, particularly for testicular biopsies where protein degradation can rapidly occur.

What are the best practices for antibody purification to enhance specificity in PCSK4 detection?

To improve specificity in PCSK4 detection through antibody purification:

• Purification methods: Evidence suggests a sequential approach yields highest specificity:

  • Protein A column purification followed by peptide affinity purification

  • Antigen affinity purification for polyclonal antibodies

• Storage considerations:

  • Store in PBS with 0.02% sodium azide and 50% glycerol pH 7.3

  • Aliquot and store at -20°C to avoid freeze/thaw cycles

  • For small (20μl) sizes, inclusion of 0.1% BSA improves stability

• Validation steps:

  • Confirm specificity through Western blotting against positive controls

  • Test serial dilutions to determine optimal concentration

  • Perform dot blot analysis - "The anti-PCSK4 molecule, which is diluted to 1:120, can still be recognized by PCSK4 antigen"

• Cross-reactivity testing: Test antibodies against related protease family members to ensure PCSK4 specificity, particularly against other proprotein convertases.

How can researchers overcome technical challenges when detecting PCSK4 in heterogeneous reproductive tissue samples?

Working with reproductive tissues presents unique challenges for PCSK4 detection:

• Sample preparation optimization:

  • For testicular tissues: Use gentle mechanical dissociation followed by enzymatic digestion to maintain protein integrity

  • For ejaculated spermatozoa: Employ density gradient centrifugation to separate mature sperm from cellular debris

  • For mixed cell populations: Consider laser capture microdissection to isolate specific cell types

• Background reduction strategies:

  • Employ extended blocking (1% BSA in PBS for 10-30 minutes)

  • Use antigen retrieval with TE buffer pH 9.0 for improved signal-to-noise ratio

  • Consider tyramide signal amplification for low-abundance detection

• Validation across different developmental stages:

  • Compare PCSK4 expression between immature and mature sperm

  • Assess different regions of the epididymis separately, as "PCSK4-like activity and CRES protein [are present] in varying levels in the fluids of various epididymal compartments"

How can PCSK4 antibodies be employed in immunocontraception research?

PCSK4 shows significant potential as an immunocontraceptive target due to its specificity to reproductive tissues. Research methodologies include:

• PCSK4 antibody development strategy:

  • Isolate PCSK4 from human spermatozoa (54 kDa band)

  • Purify using electroelution method

  • Use as immunogen in animal models

• Efficacy testing protocols:

  • In vitro: "Spermatozoa receptor binding test... using human spermatozoa, where PCSK4 54 kDa as the antigen receptor is attached to the acrosomal plasma membrane"

  • Ex vivo: "Test spermatozoa given PCSK4 antibodies" to observe reduced oocyte approach compared to controls

  • In vivo: Animal model studies testing fertility outcomes after immunization

• Specificity advantages:

  • "Sperm membrane protein has possibility to be used as candidate of male immunocontraception, because this protein only expressed in sperm membrane and not found in the other tissues"

  • "PCSK4 protein convertase enzyme is an immunogenic protein that can be used to develop a candidate male [contraceptive]"

• Methodological considerations:

  • Protein must have "molecular weight at least 10 kDa" to be sufficiently immunogenic

  • "Induction repeatedly [with] immunogen can enhance the proliferation of T cell and B cells specific to an antigen"

What are the methodological approaches for studying the regulation of PCSK4 activity by endogenous inhibitors?

Research into endogenous PCSK4 regulation provides insights into natural fertility control mechanisms:

• Inhibitor identification techniques:

  • In vitro inhibition assays using fluorogenic substrates like "Boc-RVRR-MCA"

  • Processing assays using natural substrates such as "pro-IGF-2"

• Structure-function studies:

  • Analysis of CRES (cystatin related epididymal spermatogenic) protein as a PCSK4 inhibitor

  • Comparison of inhibition potency between different oligomeric states: "while CRES-dimer exhibits Ki ~8 μM, the corresponding monomer showed Ki > 100 μM"

• Localization studies:

  • Analysis of "PC4-like activity and CRES protein in varying levels in the fluids of various epididymal compartments"

  • Correlation of inhibitor expression with PCSK4 activity along the male reproductive tract

• Physiological relevance assessment:

  • Investigation of how "regulation of sperm-PC4 activity during its storage and transport through epididymis is an important determinant for ultimate egg-binding and fertilizing capacities of sperms"

How do anti-PCSK4 antibodies differ in their reactivity across species for comparative reproductive studies?

Understanding species differences is crucial when designing comparative studies:

When designing comparative studies:

• Antibody selection: Choose antibodies raised against conserved epitopes when comparing across species

• Validation requirements: "The anti-Proprotein convertase PC4/PCSK4 antibody has not been validated for cross reactivity specifically with zebrafish tissues, though there is a good chance of cross reactivity"

• Immunogen considerations: Some antibodies are raised against species-specific sequences - "A synthetic peptide corresponding to a sequence at the N-terminus of mouse Proprotein convertase PC4, different from the related rat sequence by one amino acid"

What methodological considerations should be addressed when using PCSK4 antibodies in knockout/knockdown model systems?

When using PCSK4 antibodies in genetic modification models:

• Knockout validation strategy:

  • Confirm knockout efficiency using antibodies targeting different epitopes

  • Perform both protein (Western blot) and mRNA (qRT-PCR) validation

  • Include positive controls from wild-type tissues

• Functional compensation assessment:

  • Test for "functional redundancy among convertases" that may affect phenotype interpretation

  • Investigate whether "differential precursor cleavage due to species-specific sequence differences" affects outcomes

• Phenotypic analysis approaches:

  • "PC4 knockout mice exhibit severely impaired male fertility due to premature sperm acrosome reaction"

  • Employ multiple fertility parameters including sperm motility, acrosome reaction timing, and in vitro fertilization rates

• Tissue-specific knockdown considerations:

  • "The creation of more sophisticated mouse models with tissue- or temporally-restricted expression" may provide more precise insights

  • Compare results with "human PC polymorphisms and mutations" for translational relevance

How should researchers interpret differential PCSK4 expression patterns between normal and pathological sperm samples?

To correctly interpret differential PCSK4 expression in normal versus pathological samples:

• Quantification methods:

  • Use densitometry for Western blot band intensity measurement

  • Employ standardized scoring systems for immunohistochemistry

  • Include housekeeping protein controls for relative quantification

• Statistical analysis approaches:

  • Apply appropriate statistical tests based on data distribution

  • Consider multiple comparison corrections when analyzing various sperm parameters

  • Correlate PCSK4 expression with functional sperm parameters (motility, acrosome reaction rates)

• Confounding variables to control:

  • Account for sperm maturity and capacitation status

  • Consider patient age, hormonal status, and other medical conditions

  • Standardize sample collection and processing times

• Functional correlation:

  • Determine whether altered PCSK4 levels correlate with "acrosome reaction disturbance"

  • Assess if changes affect "penetrating the zone pellucida" ability