PRSS8 Antibody, HRP conjugated

What is PRSS8 and why is it a significant target for research?

PRSS8 (Protease, serine, 8), also known as Prostasin or Channel-activating protease 1 (CAP1), is a trypsinogen that belongs to the trypsin family of serine proteases. It is highly expressed in prostate epithelia and is one of several proteolytic enzymes found in seminal fluid. The PRSS8 proprotein undergoes cleavage to produce a light chain and a heavy chain that remain associated through a disulfide bond . This enzyme is active on peptide linkages involving the carboxyl group of lysine or arginine.

PRSS8 has significant research interest because:

• It plays a major role in regulating sodium balance and glucose homeostasis

• Aberrant PRSS8 expression has been associated with multiple cancers, including ovarian, prostate, breast, bladder, and gastric cancers

• It has been identified as a potential biomarker for early detection of ovarian cancer and Alzheimer's disease

This protein's diverse physiological roles and potential as a biomarker make HRP-conjugated antibodies against PRSS8 valuable tools for investigating its expression and function in various research contexts.

What are the key differences between PRSS8 antibodies targeting different epitopes?

PRSS8 antibodies targeting different epitopes exhibit varying specificity and application profiles depending on the amino acid sequences they recognize. Based on available antibody products, the following comparison highlights their differences:

The epitope selection affects experimental outcomes significantly. Antibodies targeting conserved regions provide cross-species reactivity, while those targeting unique epitopes offer higher specificity. Monoclonal antibodies like the F2Z4K rabbit mAb provide superior lot-to-lot consistency but may have narrower reactivity profiles compared to polyclonal antibodies .

How should I optimize HRP-conjugated PRSS8 antibody dilution for Western blotting?

Optimizing HRP-conjugated PRSS8 antibody dilution for Western blotting requires a systematic approach to achieve the optimal signal-to-noise ratio:

• Initial dilution test: Begin with the manufacturer's recommended dilution (typically 1:1000 for primary antibodies like PRSS8 F2Z4K Rabbit mAb) . Perform a gradient dilution experiment using 1:500, 1:1000, 1:2000, and 1:5000 dilutions.

• Sample preparation considerations:

  • PRSS8 has a molecular weight of approximately 36 kDa

  • Use fresh samples with protease inhibitors to prevent degradation

  • Include positive and negative control samples to verify specificity

  • Load 20-50 μg of total protein per lane

• Blocking optimization: Use 5% non-fat dry milk or 3-5% BSA in TBST. Test both if high background is observed, as PRSS8 detection may be sensitive to blocking reagent selection.

• Incubation parameters:

  • Primary antibody: Incubate at 4°C overnight for optimal sensitivity

  • Secondary antibody: Incubate for 1 hour at room temperature

  • Include sufficient washing steps (3-5 washes for 5-10 minutes each)

• Exposure time adjustment: Begin with short exposures (30 seconds) and gradually increase to avoid saturation while capturing weak signals.

The optimal dilution is one that provides specific bands at the expected molecular weight with minimal background. Typically, researchers find that 1:1000 to 1:2000 dilutions of PRSS8 antibodies yield optimal results, but this may vary based on the specific conjugate and sample type being analyzed .

What are the critical parameters for successful PRSS8 detection using ELISA with HRP-conjugated antibodies?

For successful PRSS8 detection using ELISA with HRP-conjugated antibodies, several critical parameters must be carefully controlled:

• Antibody selection: Use a matched pair of capture and detection antibodies specifically validated for PRSS8 sandwich ELISA. The Human Prostasin solid-phase sandwich ELISA method ensures exclusive recognition of both natural and recombinant human Prostasin .

• Sample preparation:

  • For serum/plasma: Dilute 1:2 to 1:10 in sample diluent to minimize matrix effects

  • For cell culture supernatant: Centrifuge at 10,000×g for 10 minutes to remove debris

  • For cell/tissue lysates: Ensure complete homogenization and clarification

• Standard curve preparation: Prepare a serial dilution of the lyophilized PRSS8 standard covering the range of 78.125-5000 pg/ml to ensure accurate quantitation .

• Assay conditions:

  • Maintain consistent temperature (room temperature, 20-25°C)

  • Adhere to precise incubation times (total assay time approximately 4 hours)

  • Use freshly prepared reagents, especially substrate solutions

• Detection and analysis:

  • Measure absorbance at 450nm with wavelength correction at 570nm

  • The absorbance value is directly proportional to PRSS8 concentration

  • Use 4-parameter logistic curve fitting for standard curve analysis

• Sensitivity considerations: The detection limit of commercially available PRSS8 ELISA kits is approximately 46.875 pg/ml , so samples with expected concentrations below this threshold may require concentration or more sensitive detection methods.

For PRSS8 ELISA, the sandwich format using double antibody methods provides superior specificity compared to competitive ELISA approaches, with no significant cross-reactivity with other analogs reported .

How should I interpret contradictory PRSS8 expression data across different detection methods?

When encountering contradictory PRSS8 expression data across different detection methods (such as Western blot, ELISA, and immunohistochemistry), a systematic troubleshooting approach is necessary:

• Antibody epitope considerations:

  • Different antibodies target distinct epitopes of PRSS8 which may be differentially accessible depending on protein conformation or processing

  • The PRSS8 proprotein is cleaved to produce light and heavy chains connected by a disulfide bond , potentially affecting epitope availability

  • Western blots performed under reducing conditions may detect different bands than under non-reducing conditions

• Post-translational modifications:

  • PRSS8 undergoes complex processing including proteolytic cleavage

  • Glycosylation states may vary between tissue types, affecting antibody recognition

  • Phosphorylation status may alter during sample processing

• Expression level quantification:

  • Compare the linear range of each detection method

  • ELISA typically has a detection range of 78.125-5000 pg/ml for PRSS8

  • Western blotting may be more suitable for relative quantification than absolute values

• Resolution strategy:

  • Create a comparative analysis table documenting all variables (sample preparation, antibodies used, detection methods)

  • Validate findings with multiple antibodies targeting different PRSS8 epitopes

  • Consider orthogonal approaches such as mass spectrometry for unbiased protein identification

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity

• Data integration approach:

  • Weight evidence based on methodological strengths (ELISA provides quantitative data, IHC provides spatial information)

  • Consider biological context (PRSS8 expression varies by tissue type)

  • Report all findings transparently, acknowledging methodological limitations

What statistical approaches are most appropriate for analyzing PRSS8 expression differences between experimental groups?

When analyzing PRSS8 expression differences between experimental groups, selecting appropriate statistical methods is crucial for robust data interpretation:

• For normally distributed data with equal variances:

  • Student's t-test for comparing two groups

  • One-way ANOVA followed by Tukey's or Bonferroni post-hoc tests for multiple group comparisons

  • Two-way ANOVA for examining the effects of two independent variables

• For non-normally distributed data or heterogeneous variances:

  • Mann-Whitney U test for two-group comparisons

  • Kruskal-Wallis test followed by Dunn's post-hoc test for multiple groups

  • Permutation tests for small sample sizes

• For time-course or repeated measures experiments:

  • Repeated measures ANOVA if assumptions are met

  • Mixed-effects models to account for missing data points

  • Time series analysis for detailed temporal patterns

• Power analysis considerations:

  • Based on published PRSS8 expression studies, a minimum sample size of 6-8 per group is typically needed to detect a 50% difference in expression with 80% power at α=0.05

  • Higher sample sizes may be required for detecting subtle changes in PRSS8 expression

• Correlation analysis with clinical parameters:

  • Pearson correlation for linear relationships with normally distributed variables

  • Spearman rank correlation for non-parametric data

  • Multiple regression to identify independent predictors of PRSS8 expression

• Visualization approaches:

  • Box plots showing median, interquartile range, and outliers

  • Violin plots to visualize distribution characteristics

  • Forest plots for meta-analysis of PRSS8 expression across studies

• Considerations for ELISA data specifically:

  • Transform data to address heteroscedasticity (common in ELISA)

  • Use weighted regression for standard curves

  • Apply four-parameter logistic regression for accurate concentration determination

For publicly reported studies on PRSS8 expression in cancer tissues, significance levels of p<0.05 after appropriate multiple testing corrections are generally considered statistically meaningful, with fold changes of >1.5 typically representing biological significance.

How can PRSS8 antibody-based techniques be optimized for detecting low-abundance PRSS8 in extracellular vesicles?

Detecting low-abundance PRSS8 in extracellular vesicles (EVs) requires specialized optimization of antibody-based techniques:

• Sample enrichment strategies:

  • Differential ultracentrifugation with 100,000-200,000×g spin for 2-4 hours

  • Size exclusion chromatography to purify EV populations

  • Immunoaffinity capture using EV marker antibodies (CD63, CD9, CD81)

  • Polymer-based precipitation followed by washing to concentrate EV proteins

• Signal amplification approaches:

  • Tyramide signal amplification (TSA) for enhanced HRP catalytic activity

  • Poly-HRP conjugated secondary antibodies providing 3-5× signal enhancement

  • Proximity ligation assay (PLA) for single-molecule detection sensitivity

  • Microfluidic-based digital ELISA for sub-pg/ml detection limits

• Detection optimization:

  • Extended substrate incubation times (up to 30 minutes) with temperature control

  • Use of supersensitive chemiluminescent substrates with enhanced light output

  • Cooled CCD camera imaging for Western blots to improve signal collection

  • Narrow bandwidth filters for fluorescent detection to reduce background

• Multiplex strategies:

  • Co-localization of PRSS8 with EV markers using dual-color immunofluorescence

  • Sequential probing of multiple PRSS8 epitopes to confirm true positives

  • Combination of PRSS8 antibody detection with proteomic MS/MS validation

• Controls and validation:

  • Include synthetic PRSS8 peptide standards spiked into negative samples

  • Use EVs from PRSS8-knockout and PRSS8-overexpressing cell lines

  • Compare EV samples with corresponding cell lysates for expression patterns

The sensitivity threshold can be pushed to detect PRSS8 concentrations below 50 pg/ml by implementing these optimizations collectively . For maximum sensitivity, researchers should consider using monoclonal antibodies like PRSS8 F2Z4K that have been validated for detecting endogenous levels of the protein .

What are the current methodological approaches for investigating PRSS8's role in cancer progression using antibody-based techniques?

Current methodological approaches for investigating PRSS8's role in cancer progression using antibody-based techniques span multiple experimental paradigms:

• Tissue microarray (TMA) analysis:

  • Correlation of PRSS8 expression with tumor stage, grade, and patient outcomes

  • Multiplexed IHC to examine PRSS8 co-localization with cancer markers

  • Quantitative image analysis using digital pathology platforms

  • Example research finding: PRSS8 expression was reported to correlate inversely with tumor grade in ovarian cancer, suggesting its potential as a prognostic biomarker

• Cell-based functional assays:

  • Antibody-mediated neutralization of PRSS8 in cell culture models

  • Assessment of migration, invasion, and proliferation after PRSS8 inhibition

  • Monitoring of epithelial-mesenchymal transition (EMT) markers in relation to PRSS8

  • Co-immunoprecipitation to identify PRSS8 protein interaction partners

• Signaling pathway analysis:

  • Phospho-specific antibody arrays to identify PRSS8-dependent signaling changes

  • Western blot analysis of canonical pathways (MAPK, PI3K/AKT) following PRSS8 modulation

  • ChIP-seq to identify transcriptional targets regulated by PRSS8-dependent signaling

• In vivo modeling approaches:

  • Intravital microscopy with fluorescently labeled PRSS8 antibodies

  • Xenograft models with PRSS8 knockdown/overexpression

  • PRSS8 antibody-drug conjugates for targeted therapy assessment

  • Single-cell analysis of tumor heterogeneity in PRSS8 expression

• Liquid biopsy applications:

  • Development of circulating PRSS8 detection as a minimally invasive biomarker

  • Exosomal PRSS8 quantification using sandwich ELISA with 46.875 pg/ml sensitivity

  • Correlation of serum PRSS8 levels with tissue expression and clinical outcomes

• Translational research applications:

  • PRSS8 antibody-based companion diagnostics for patient stratification

  • Multiplex biomarker panels including PRSS8 for improved diagnostic accuracy

  • Development of tissue-based prognostic algorithms incorporating PRSS8 expression

These methodological approaches have revealed that PRSS8 may function as either a tumor suppressor or oncogene depending on cancer type and context, highlighting the need for cancer-specific experimental designs when studying this protein.

What strategies can address non-specific binding and background issues when using HRP-conjugated PRSS8 antibodies?

Non-specific binding and background issues with HRP-conjugated PRSS8 antibodies can significantly impact experimental results. The following systematic troubleshooting strategies address these common challenges:

• Blocking optimization:

  • Test different blocking agents: 5% non-fat dry milk, 3-5% BSA, commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

  • Consider species-specific normal serum (1-5%) matching the host of secondary antibody

• Antibody dilution and incubation conditions:

  • Increase antibody dilution incrementally (try 1:2000 to 1:5000)

  • Prepare antibodies in blocking buffer containing 0.05-0.1% Tween-20

  • Switch from room temperature to 4°C incubation for primary antibody

  • Use antibody diluent containing 0.1-0.5% BSA and 0.05% sodium azide for stability

• Washing optimization:

  • Increase washing frequency (5-6 times) and duration (10 minutes per wash)

  • Use higher concentration of Tween-20 (0.1-0.2%) in wash buffer

  • Include one wash with high salt buffer (500mM NaCl) to disrupt low-affinity interactions

  • Use gentle agitation during washing steps

• Sample-specific considerations:

  • Pre-absorb antibody with proteins from the sample species

  • Treat samples with commercially available background reducers

  • Use protein A/G pre-clearing of lysates to remove sticky proteins

  • Include additional protease inhibitors to prevent epitope degradation

• HRP-specific optimizations:

  • Use fresh substrate prepared immediately before use

  • Reduce substrate incubation time if background develops quickly

  • Consider switching to fluorescent detection methods if HRP background persists

  • Use HRP inhibitors like sodium azide during antibody storage but not during detection

• Control experiments:

  • Include no-primary antibody control to identify secondary antibody background

  • Use isotype control antibodies at the same concentration

  • Include PRSS8-negative tissue or knockdown cell lines as biological controls

  • Perform peptide competition assays with the immunizing peptide for antibody validation

For PRSS8-specific considerations, remember that the protein undergoes post-translational modifications and cleavage, which may affect antibody specificity. Using monoclonal antibodies like the F2Z4K clone can help reduce non-specific binding compared to polyclonal alternatives .

How can researchers validate the specificity of PRSS8 antibodies for their particular experimental system?

Thorough validation of PRSS8 antibody specificity for each experimental system is essential for generating reliable research data. A comprehensive validation approach includes:

• Genetic manipulation controls:

  • CRISPR/Cas9 knockout of PRSS8 in relevant cell lines

  • siRNA or shRNA knockdown with 48-72 hour post-transfection testing

  • Overexpression of tagged PRSS8 constructs for co-localization studies

  • Rescue experiments reintroducing PRSS8 in knockout backgrounds

• Multiple antibody comparison:

  • Test antibodies targeting different PRSS8 epitopes (e.g., AA 33-218, AA 65-165, Val98 region)

  • Compare monoclonal (e.g., F2Z4K) versus polyclonal antibodies

  • Cross-validate with commercially available antibodies from different vendors

  • Verify consistent protein size detection (~36 kDa) across antibody types

• Cross-reactivity assessment:

  • Test antibody in species with known PRSS8 sequence homology

  • Examine reactivity in tissues with varying levels of PRSS8 expression

  • Check for cross-reactivity with related proteases using recombinant proteins

  • Perform peptide competition assays with specific and non-specific peptides

• Orthogonal method validation:

  • Correlate protein expression with mRNA levels by RT-qPCR

  • Compare antibody-based detection with mass spectrometry results

  • Verify subcellular localization using fractionation followed by Western blotting

  • Confirm functional activity using PRSS8 enzyme activity assays

• Application-specific validation:

  • For IHC: Include antigen retrieval optimization and concentration gradients

  • For Western blotting: Test reducing vs. non-reducing conditions

  • For ELISA: Perform spike-and-recovery experiments with recombinant PRSS8

  • For IP: Verify enrichment by comparing input, flow-through, and elution fractions

• Documentation and reporting standards:

  • Record complete antibody metadata (catalog #, lot #, dilution, incubation conditions)

  • Include all validation data in publications or supplementary materials

  • Report concordant and discordant findings with transparent discussion

  • Share validation protocols through repositories or protocol-sharing platforms

This systematic validation ensures that observed signals genuinely represent PRSS8 rather than artifacts or cross-reactivity, particularly important given that PRSS8 shares structural features with other serine proteases that could potentially lead to false positive results.

How are HRP-conjugated PRSS8 antibodies being utilized in current Alzheimer's disease biomarker research?

Recent investigations into PRSS8 as a potential biomarker for Alzheimer's disease (AD) have employed HRP-conjugated antibodies in several innovative approaches:

• CSF and blood-based biomarker development:

  • Sandwich ELISA systems with sensitivity thresholds of ~46.875 pg/ml are being optimized for detecting PRSS8 in cerebrospinal fluid and plasma samples

  • Correlative studies examining relationships between PRSS8 levels and established AD biomarkers (Aβ42, tau, p-tau)

  • Longitudinal measurements in preclinical and prodromal AD patients to assess prognostic value

  • Development of multiplexed assays incorporating PRSS8 alongside traditional AD biomarkers

• Neuronal tissue analysis:

  • Immunohistochemical investigations of PRSS8 distribution in post-mortem brain tissues from AD patients versus age-matched controls

  • Co-localization studies with amyloid plaques and neurofibrillary tangles

  • Quantitative region-specific expression analysis across Braak stages

  • Comparison of neuronal versus glial PRSS8 expression patterns in disease progression

• Mechanistic investigations:

  • Analysis of PRSS8's proteolytic activity on AD-related substrates

  • Evaluation of PRSS8's role in regulating neuroinflammatory processes

  • Investigation of interactions between PRSS8 and membrane ion channels in neuronal function

  • Assessment of potential relationships between PRSS8 and blood-brain barrier integrity

• Technological adaptations:

  • Development of single-molecule array (Simoa) ultra-sensitive assays for PRSS8 detection

  • Implementation of automated ELISA platforms for high-throughput PRSS8 screening

  • Integration of PRSS8 detection into multiparametric flow cytometry panels for immune cell analysis

  • Adaptation of PRSS8 antibodies for PET imaging agent development

Preliminary research findings suggest potential correlations between altered PRSS8 levels and AD pathology, with ongoing studies working to establish whether these changes precede clinical symptoms or represent downstream effects of disease processes . The field is still emerging, with researchers actively working to determine whether PRSS8 represents a causative factor, compensatory response, or bystander in AD pathophysiology.

What are the technical considerations for developing multiplex assays that include PRSS8 detection alongside other biomarkers?

Developing multiplex assays that include PRSS8 alongside other biomarkers presents several technical challenges that require careful consideration:

• Antibody compatibility assessment:

  • Cross-reactivity testing between all antibody pairs in the multiplex panel

  • Optimization of antibody concentrations to achieve balanced signal intensity across markers

  • Evaluation of capture antibody stability when co-immobilized with other antibodies

  • Testing for competitive binding effects when multiple targets are present

• Dynamic range harmonization:

  • PRSS8 ELISA assays typically have a detection range of 78.125-5000 pg/ml

  • Adjustment of individual biomarker sensitivity to accommodate concentration differences

  • Implementation of multi-step dilution protocols for samples with widely varying analyte concentrations

  • Development of algorithms to extrapolate concentrations outside the linear range

• Signal segregation methods:

  • Spectral separation when using multiple fluorophores (minimum 30nm wavelength difference)

  • Spatial separation using microarray or microfluidic compartmentalization

  • Temporal separation using time-resolved fluorescence

  • Barcoded particles (beads) for simultaneous detection of multiple analytes

• Buffer and reagent optimization:

  • Identification of universal assay buffers compatible with all antibody-antigen interactions

  • Testing for additive effects of blocking reagents on multiple targets

  • Evaluation of detergent concentrations that balance background reduction with epitope preservation

  • Development of universal wash protocols that maintain specific binding across all targets

• Cross-platform validation:

  • Correlation of multiplex results with single-plex assays for each biomarker

  • Establishment of normalization factors for platform-specific signal differences

  • Spike-in recovery testing across a concentration gradient for all analytes

  • Evaluation of matrix effects on multiplex performance

• Data analysis considerations:

  • Implementation of multi-parametric algorithms for pattern recognition

  • Development of standardized reference materials for inter-laboratory comparison

  • Establishment of normal reference ranges for biomarker combinations

  • Integration of machine learning approaches for complex biomarker signature analysis