PCSK2 Antibody, HRP conjugated

What is PCSK2 and what are its biological functions?

PCSK2, also known as Neuroendocrine convertase 2 (NEC 2), is a serine endopeptidase belonging to the proprotein convertase family. It plays a crucial role in the processing of hormone and protein precursors at sites comprised of pairs of basic amino acid residues. PCSK2 is particularly important for the release of glucagon from proglucagon in pancreatic A cells . As a key enzyme in neuroendocrine pathways, PCSK2 is responsible for converting inactive prohormones into their biologically active forms, making it essential for various physiological processes including glucose homeostasis and neuropeptide signaling. The enzyme has an EC designation of 3.4.21.94 and is sometimes referred to as KEX2-like endoprotease 2 . PCSK2's activity is primarily localized to secretory vesicles in neuroendocrine cells, where it encounters its prohormone substrates during the regulated secretory pathway.

What are the key characteristics of PCSK2 antibodies used in research?

PCSK2 antibodies come in various forms to suit different experimental applications. The HRP-conjugated PCSK2 antibody (such as product A30790) is a polyclonal antibody raised in rabbits against recombinant rat Neuroendocrine convertase 2 protein (residues 109-637AA) . This antibody is specifically reactive with rat PCSK2 and has been validated for ELISA applications . Its polyclonal nature provides multiple epitope recognition, potentially increasing detection sensitivity. The antibody is supplied in a liquid form with a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . Alternative forms include recombinant monoclonal antibodies like the anti-PCSK2 antibody [EPR23578-19], which has broader applications including Western blot, flow cytometry, immunohistochemistry, and multiplex immunohistochemistry, with reactivity against human samples .

What is the expected molecular weight of PCSK2 in Western blot analysis?

When performing Western blot analysis with PCSK2 antibodies, researchers should expect to detect a band at approximately 70-71 kDa, which corresponds to the pro-PCSK2 form of the protein . This observation is consistent with published literature (as referenced in PMIDs 8557169 and 14693708) . In experimental validations, Western blots using PCSK2 antibodies consistently show bands at approximately 70 kDa in various cell lysates including human thyroid carcinoma epithelial cells (TT) , as well as MCF-7 human breast cancer cells, beta TC-6 mouse beta cell insulinoma cells, and HepG2 human hepatocellular carcinoma cells . It's important to note that the predicted band size is 71 kDa, while the observed band size is typically around 70 kDa . This slight discrepancy is normal and reflects the typical margin of error in molecular weight estimation on SDS-PAGE gels.

Which tissues and cell types are known to express PCSK2?

PCSK2 expression has been documented in several tissues and cell types, primarily those with neuroendocrine functions. Immunohistochemical analyses have revealed positive staining in human adrenal gland, particularly in the adrenal medulla . PCSK2 is also prominently expressed in myenteric neurons of human colon and human pancreatic islets . The enzyme has been detected in various cell lines including TT (human thyroid carcinoma epithelial cells), MCF-7 (human breast cancer cells), HepG2 (human hepatocellular carcinoma cells), and beta TC-6 (mouse beta cell insulinoma cells) . This expression pattern aligns with PCSK2's role in prohormone processing within the neuroendocrine system. Multiplex immunohistochemistry studies have shown that PCSK2 co-localizes with neuropeptide Y in chromaffin cells of the adrenal medulla, further supporting its neuroendocrine function .

How should I optimize antibody dilutions for different applications?

Optimizing antibody dilutions is critical for obtaining specific signals while minimizing background. For PCSK2 antibodies, the optimal dilution varies by application:

For Western blot applications:

• Anti-PCSK2 antibody [EPR23578-19] has been successfully used at 1/1000 dilution for human thyroid carcinoma epithelial cell lysates

• Sheep Anti-Human PCSK2 antibody has shown good results at 1 μg/mL concentration

• Sample loading of approximately 40 μg of whole cell lysate is recommended

For immunohistochemistry applications:

• A dilution of 1/2000 (0.263 μg/ml) has been effective for paraffin-embedded tissues

• Incubation for 30 minutes at room temperature has yielded good results

For immunofluorescence:

• 10 μg/mL concentration with 3-hour room temperature incubation has been reported for successful staining

For flow cytometry:

• A dilution of 1/500 (0.1μg) has been effective for fixed and permeabilized cells

Optimization should follow a systematic approach starting with the manufacturer's recommended dilution, followed by a dilution series to identify the optimal signal-to-noise ratio. Always include appropriate positive and negative controls to accurately assess staining specificity at each dilution tested.

What are the critical parameters for successful multiplex immunohistochemistry with PCSK2 antibodies?

Multiplex immunohistochemistry (mIHC) with PCSK2 antibodies requires careful consideration of several critical parameters to achieve successful results:

• Antibody compatibility: PCSK2 antibodies have been successfully used in multiplex panels with antibodies against C11B2/CYP11B2, SULT2A1, CYP11A1, Collagen VI, and Neuropeptide Y . Ensure antibodies are raised in different host species or are of different isotypes to prevent cross-reactivity.

• Optimized dilutions: For mIHC applications, PCSK2 antibody [EPR23578-19] has been effectively used at a dilution of 1:2000 (0.263 μg/ml) .

• Staining sequence: Sequential staining protocols have shown good results, with PCSK2 antibody typically applied in the first round of staining followed by other antibodies .

• Antigen retrieval: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes has proven effective for PCSK2 detection in multiplex panels .

• Detection system: Tyramide signal amplification systems with distinct fluorophores for each antibody provide optimal results for multiplexing. Each antibody incubation should be followed by a separate fluorescent tyramide signal amplification step .

• Imaging parameters: Confocal microscopy (e.g., Leica SP8) has been used for capturing high-quality multiplex images with appropriate spectral separation between fluorophores .

These parameters have been validated in studies examining PCSK2 expression in adrenal gland tissues, where PCSK2 was successfully co-stained with markers of different adrenal zones and cell types .

How can I validate the specificity of PCSK2 antibody staining?

Validating the specificity of PCSK2 antibody staining is crucial for ensuring reliable research results. A comprehensive validation approach should include:

• Positive and negative tissue controls: Use tissues with known PCSK2 expression (e.g., adrenal medulla, pancreatic islets, colon myenteric neurons) as positive controls . Tissues known to lack PCSK2 expression serve as biological negative controls.

• Technical controls: Include a "secondary antibody only" control to assess non-specific binding of the detection system. This approach has been documented using rabbit-specific IHC polymer detection kit HRP/DAB (ab209101) .

• Isotype controls: Use a non-specific antibody of the same isotype and concentration as the PCSK2 antibody. For example, rabbit monoclonal IgG (ab172730) has been used as an isotype control for rabbit anti-PCSK2 antibodies in flow cytometry applications .

• Peptide competition assays: Pre-incubate the PCSK2 antibody with excess immunizing peptide before application to the sample. Specific staining should be abolished or significantly reduced.

• Genetic validation: Compare staining between wild-type samples and those with PCSK2 knockdown or knockout. This provides the most stringent specificity control.

• Cross-validation with other techniques: Confirm immunostaining results with orthogonal methods such as Western blot or qPCR to verify PCSK2 expression in the tissues or cells being studied.

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of PCSK2 and compare staining patterns. Consistent results increase confidence in specificity.

What critical factors affect Western blot detection of PCSK2?

Successful Western blot detection of PCSK2 depends on several critical factors:

• Sample preparation: Complete cell lysis with appropriate buffers containing protease inhibitors is essential. PCSK2 is primarily localized in secretory vesicles, which may require specialized lysis conditions. A sample loading of 40 μg of whole cell lysate has been effective in published protocols .

• Denaturation conditions: Ensure complete protein denaturation by heating samples at 95°C for 5 minutes in reducing buffer. The observed 70 kDa pro-PC2 band is detected under reducing conditions .

• Gel percentage and transfer parameters: Use 8-10% gels for optimal resolution of the 70 kDa PCSK2 protein. Transfer conditions may need optimization, with wet transfer potentially providing better results for this molecular weight range.

• Blocking conditions: 5% non-fat dry milk in TBST has been successfully used as a blocking buffer . Optimize blocking time to prevent over-blocking while minimizing background.

• Antibody incubation: Successful protocols have used 1/1000 dilution of anti-PCSK2 antibody [EPR23578-19] or 1 μg/mL of sheep anti-human PCSK2 antibody . Primary antibody incubation overnight at 4°C may improve signal quality.

• Detection system sensitivity: Enhanced chemiluminescence detection with exposure time of approximately 37 seconds has been reported as effective . HRP-conjugated secondary antibodies such as Goat Anti-Rabbit IgG H&L (HRP) at 1/50000 dilution have shown good results .

• Positive controls: Include lysates from cells known to express PCSK2, such as TT, MCF-7, HepG2, or beta TC-6 cells , to confirm antibody performance and proper experimental conditions.

What is the optimal protocol for using HRP-conjugated PCSK2 antibodies in ELISA?

The HRP-conjugated PCSK2 polyclonal antibody is particularly suitable for ELISA applications . An optimized protocol should include:

• Plate preparation: Coat high-binding 96-well plates with capture antibody or antigen (depending on ELISA format) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or non-fat dry milk in PBS for 1-2 hours at room temperature.

• Sample preparation: Prepare cell or tissue lysates using appropriate lysis buffers containing protease inhibitors. For serum/plasma samples, dilute appropriately based on expected PCSK2 concentration.

• Antibody dilution: Prepare the HRP-conjugated PCSK2 antibody in blocking buffer. Start with the manufacturer's recommended dilution and optimize as needed.

• Incubation parameters: Add samples and antibodies to appropriate wells and incubate at room temperature for 1-2 hours or at 4°C overnight. Maintain consistent timing between wells for reproducible results.

• Washing steps: Perform 4-6 washes with PBS containing 0.05-0.1% Tween-20 after each incubation step to remove unbound reagents.

• Detection method: Add TMB substrate solution and incubate for 15-30 minutes at room temperature protected from light. Stop the reaction with 2N H₂SO₄ and read absorbance at 450nm.

• Quality controls: Include standard curves using recombinant PCSK2, positive and negative controls, and blank wells to ensure assay validity and enable quantitative analysis.

Given the 50% glycerol content in the antibody buffer , ensure proper dilution to prevent interference with coating or detection steps.

How can I effectively use PCSK2 antibodies for immunofluorescence staining?

Successful immunofluorescence staining with PCSK2 antibodies requires attention to several critical steps:

• Cell preparation: Culture cells on appropriate coverslips or chamber slides at 70-80% confluence. Use cell lines with known PCSK2 expression such as HepG2, which has been successfully used in published protocols .

• Fixation method: Immersion fixation has been successfully employed for PCSK2 staining . For most applications, 4% paraformaldehyde in PBS for 10-15 minutes at room temperature provides good epitope preservation.

• Permeabilization: For formaldehyde-fixed cells, permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to enable antibody access to intracellular PCSK2.

• Blocking step: Block with 1-5% normal serum (matched to the secondary antibody host species) with 0.1-0.3% Triton X-100 in PBS for 30-60 minutes at room temperature.

• Primary antibody incubation: Published protocols have used sheep anti-human/mouse PCSK2 antibody at 10 μg/mL concentration for 3 hours at room temperature . Dilute antibody in blocking solution.

• Secondary antibody selection: Use fluorophore-conjugated secondary antibody matching the host species of the primary antibody. NorthernLights™ 557-conjugated Anti-Sheep IgG has been successfully used with sheep primary antibodies at appropriate dilutions .

• Nuclear counterstaining: DAPI has been effectively used as a nuclear counterstain in PCSK2 immunofluorescence protocols .

• Mounting and imaging: Mount with anti-fade mounting medium and seal edges for long-term preservation. Image using appropriate filter sets and exposure settings to maximize signal-to-noise ratio.

This approach has successfully visualized PCSK2 in HepG2 human hepatocellular carcinoma cells , providing valuable information about its subcellular localization.

What protocols are recommended for using PCSK2 antibodies in Western blot applications?

For optimal Western blot detection of PCSK2, the following protocol has been validated in published research:

• Sample preparation:

  • Lyse cells in appropriate buffer containing protease inhibitors

  • Quantify protein concentration using a reliable method

  • Prepare samples containing 40 μg of whole cell lysate per lane

  • Add reducing sample buffer and heat at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Separate proteins on SDS-PAGE (8-10% gel recommended for 70 kDa PCSK2)

  • Transfer to PVDF membrane using standard protocols

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

  • Block membrane with 5% non-fat dry milk in TBST

  • Block for 1 hour at room temperature with gentle agitation

• Primary antibody incubation:

  • Dilute anti-PCSK2 antibody at appropriate concentration

    • 1/1000 dilution for anti-PCSK2 antibody [EPR23578-19]

    • 1 μg/mL for sheep anti-human PCSK2 antibody

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash 3-5 times with TBST, 5 minutes each

• Secondary antibody incubation:

  • For rabbit primary antibodies: Goat Anti-Rabbit IgG H&L (HRP) at 1/50000 dilution

  • For sheep primary antibodies: HRP-conjugated Anti-Sheep IgG at appropriate dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Apply enhanced chemiluminescence substrate according to manufacturer's instructions

  • Exposure time of approximately 37 seconds has been effective

  • Capture images using digital imaging system

• Controls:

  • Include positive control lysates from cells known to express PCSK2 (TT, MCF-7, HepG2, or beta TC-6 cells)

  • Include molecular weight markers to confirm the 70 kDa band size

This protocol has successfully detected the 70 kDa pro-PC2 form in multiple experimental systems .

How should I store and handle HRP-conjugated PCSK2 antibodies to maintain activity?

Proper storage and handling of HRP-conjugated PCSK2 antibodies is essential for maintaining their activity and ensuring consistent experimental results:

• Long-term storage:

  • Upon receipt, store at -20°C or -80°C as recommended by the manufacturer

  • Avoid repeated freeze-thaw cycles that can degrade both the antibody and the HRP conjugate

• Aliquoting strategy:

  • Divide stock antibody into small, single-use aliquots

  • Use sterile tubes and aseptic technique to prevent contamination

  • Record the date of aliquoting on each tube

• Buffer considerations:

  • The documented buffer composition includes 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative

  • Do not add sodium azide as it inhibits HRP activity

  • High glycerol concentration (50%) helps maintain antibody stability during freezing

• Working solution stability:

  • Prepare working dilutions fresh before each experiment

  • If storage of diluted antibody is necessary, keep at 4°C for no more than 1-2 weeks

  • Always centrifuge briefly before use to collect all liquid at the bottom of the tube

• Handling precautions:

  • Minimize exposure to light which can reduce HRP activity

  • Avoid contamination with heavy metals or oxidizing agents

  • Keep on ice when using during experiments

  • Return to proper storage temperature promptly after use

• Quality monitoring:

  • Include positive controls in each experiment to track antibody performance over time

  • Consider purchasing a new lot if signal quality diminishes despite optimal experimental conditions

Adhering to these storage and handling guidelines will help maintain antibody specificity and HRP enzymatic activity, ensuring consistent and reliable experimental results.

How should I quantify PCSK2 levels in Western blot experiments?

Accurate quantification of PCSK2 in Western blot experiments requires careful attention to several key factors:

• Image acquisition:

  • Capture digital images using a CCD camera-based system

  • Ensure signal is within the linear range of detection (not saturated)

  • Documented exposure time of 37 seconds has been effective for PCSK2 Western blots

  • Capture multiple exposures if unsure about linear range

• Background subtraction:

  • Define background regions for consistent subtraction

  • Use lane-specific or global background correction depending on blot quality

  • Ensure background regions are representative of actual background

• Normalization approach:

  • Normalize PCSK2 signal (70 kDa band) to loading controls

  • Suitable loading controls include β-actin, GAPDH, or total protein stains

  • Ensure loading control expression is stable across experimental conditions

  • Consider using total protein normalization (e.g., Ponceau S or stain-free technology) for more reliable quantification

• Software tools:

  • Use dedicated image analysis software (e.g., ImageJ, Image Studio)

  • Apply consistent analysis settings across all blots in a study

  • Define bands using rectangular or freehand selection tools

  • Extract integrated density values rather than peak intensity

• Statistical analysis:

  • Perform experiments in biological triplicates at minimum

  • Apply appropriate statistical tests based on experimental design

  • Present data as fold-change relative to control or absolute values if standards are available

  • Report means with measures of variability (standard deviation or standard error)

• Data presentation:

  • Include representative blot images alongside quantification graphs

  • Show molecular weight markers on blot images

  • Indicate specific band being quantified (70 kDa pro-PCSK2)

  • Present normalized values in graphical format with appropriate statistics

This quantification approach has been validated in studies examining PCSK2 expression in various cell types including TT, MCF-7, HepG2, and beta TC-6 cells .

How do I interpret variations in PCSK2 band patterns in Western blots?

Variations in PCSK2 band patterns in Western blots can provide valuable information about protein processing, modification states, or potential degradation. Proper interpretation requires understanding several factors:

• Expected pattern:

  • The primary PCSK2 band is observed at approximately 70 kDa, representing pro-PCSK2

  • This molecular weight is consistent with published literature references (PMID:8557169, 14693708)

• Multiple band interpretation:

  • Additional lower molecular weight bands may represent processed forms of PCSK2

  • PCSK2 undergoes proteolytic processing from its precursor form to generate the mature enzyme

  • Upper molecular weight bands may indicate post-translational modifications or aggregation

• Tissue-specific processing:

  • Different tissues may show varying ratios of precursor to mature forms

  • Compare your results with published data from similar experimental systems

  • Consider the physiological state of the tissue (e.g., stimulated vs. basal conditions)

• Technical considerations:

  • Sample preparation can affect band patterns (protease activity during extraction)

  • Loading too much protein can cause smearing or aberrant migration

  • Incomplete reduction can result in higher molecular weight bands

  • Degradation during storage may generate lower molecular weight fragments

• Validation approaches:

  • If unexpected bands appear, validate with alternative antibodies

  • Compare with recombinant PCSK2 standard if available

  • Consider immunoprecipitation followed by Western blot to confirm specificity

  • For definitive identification, use immunoprecipitation followed by mass spectrometry

• Comparison with positive controls:

  • Include lysates from cells with known PCSK2 expression patterns such as TT cells, MCF-7, HepG2, and beta TC-6 cells

  • Compare band patterns between your samples and these established controls

What approaches are recommended for comparing PCSK2 expression across different experimental models?

When comparing PCSK2 expression across different experimental models, several methodological considerations are critical for valid comparisons:

These approaches have been successfully employed in studies comparing PCSK2 expression across different cell lines including MCF-7, beta TC-6, and HepG2 cells .

How can I design experiments to study PCSK2's role in hormone processing?

Designing experiments to investigate PCSK2's role in hormone processing requires a comprehensive approach:

• Model system selection:

  • Choose models with endogenous PCSK2 expression

  • Pancreatic islets or cell lines (e.g., beta TC-6) for studying glucagon processing

  • Adrenal gland or adrenal cell lines for neuropeptide processing

  • Human cell lines such as TT (thyroid carcinoma), MCF-7 (breast cancer), or HepG2 (hepatocellular carcinoma) have demonstrated PCSK2 expression

• Genetic manipulation strategies:

  • RNA interference (siRNA/shRNA) for transient PCSK2 knockdown

  • CRISPR-Cas9 for generating stable PCSK2 knockout cell lines

  • Overexpression studies using wild-type and mutant PCSK2 constructs

  • Rescue experiments to confirm phenotype specificity

• Analytical approaches:

  • Western blot analysis to detect:

    • PCSK2 expression (70 kDa band)

    • Prohormone substrates and their processed products

  • Immunofluorescence to assess:

    • PCSK2 subcellular localization

    • Co-localization with hormone precursors

  • Mass spectrometry to:

    • Identify specific cleavage sites

    • Discover novel PCSK2 substrates

    • Quantify peptide products

• Functional assays:

  • Enzyme activity assays using fluorogenic substrates

  • ELISA to quantify processed hormone levels

  • Secretion assays to measure regulated hormone release

  • Cell-based assays to assess biological activity of processed hormones

• In vivo validation:

  • Animal models with PCSK2 modulation

  • Tissue-specific manipulation using Cre-loxP systems

  • Phenotypic analysis focused on endocrine parameters

  • Physiological challenges to reveal subtle phenotypes

• Controls and validation:

  • Include positive controls (tissues/cells with known PCSK2 function)

  • Employ multiple approaches to confirm findings

  • Validate antibody specificity for all applications

  • Use appropriate statistical analysis for experimental design

This multi-faceted approach has been successfully employed to study PCSK2's role in various neuroendocrine tissues, including pancreatic islets and adrenal medulla .

What experimental approaches are suitable for investigating PCSK2 in multiplex studies?

Multiplex studies investigating PCSK2 alongside other proteins provide valuable insights into complex biological systems. Several experimental approaches have been validated:

• Multiplex immunohistochemistry (mIHC):

  • PCSK2 antibodies have been successfully incorporated into multiplex panels with:

    • Adrenal cortex markers: C11B2/CYP11B2, SULT2A1, CYP11A1

    • Structural proteins: Collagen VI

    • Neuroendocrine markers: Neuropeptide Y

  • A dilution of 1:2000 (0.263 μg/ml) has been effective for PCSK2 antibody in mIHC applications

  • Sequential staining protocols work well, with PCSK2 typically applied in the first round

  • Tyramide signal amplification systems with distinct fluorophores enable clear discrimination between markers

• Multiplexed Western blot analysis:

  • Sequential probing of the same membrane for PCSK2 and other proteins

  • Stripping and reprobing techniques with thorough validation

  • Multiplex fluorescent Western blot using different fluorophore-conjugated secondary antibodies

  • Appropriate size separation should be ensured if targeting multiple proteins

• Flow cytometry:

  • PCSK2 has been successfully detected by flow cytometry in fixed and permeabilized cells

  • A dilution of 1/500 (0.1μg) has been effective

  • Can be combined with surface markers and other intracellular proteins

  • Proper compensation controls are essential for accurate multiplexing

• Bead-based multiplex assays:

  • Development of multiplex bead arrays for PCSK2 and related proteins

  • Enables simultaneous quantification of multiple analytes

  • Requires thorough validation for antibody specificity and lack of cross-reactivity

• Mass spectrometry-based approaches:

  • Targeted multiple reaction monitoring (MRM) assays

  • Parallel reaction monitoring (PRM) for improved specificity

  • Label-free or isobaric labeling for relative quantification

  • Absolute quantification using isotope-labeled standards

• Spatial transcriptomics combined with protein detection:

  • In situ sequencing or spatial transcriptomics to detect PCSK2 mRNA

  • Combined with protein detection for multi-omics spatial analysis

  • Provides insights into transcriptional and translational regulation

These approaches enable comprehensive analysis of PCSK2 in the context of broader biological systems, as demonstrated in studies of adrenal gland where PCSK2 co-localization with various markers provided insights into its tissue-specific functions .

How can I design experiments to validate PCSK2 antibody specificity across species?

Validating PCSK2 antibody specificity across species is crucial for comparative studies. A comprehensive validation strategy should include:

• Sequence homology analysis:

  • Compare PCSK2 protein sequences across target species

  • Identify the specific epitope recognized by the antibody

  • Assess epitope conservation across species

  • For example, while some antibodies are specific to rat PCSK2 , others recognize human and mouse PCSK2

• Western blot validation:

  • Test the antibody against samples from each target species

  • Include positive controls from species with confirmed reactivity

  • Compare band patterns and molecular weights

  • Published data shows consistent detection of approximately 70 kDa PCSK2 bands in both human and mouse samples

• Peptide competition assays:

  • Perform peptide blocking using the immunizing peptide

  • Test blocking efficiency across samples from different species

  • Specific binding should be abolished or significantly reduced in all target species

• Genetic validation approaches:

  • Test antibody on samples from PCSK2 knockout models

  • Use siRNA knockdown in cells from different species

  • Compare staining patterns between wild-type and knockdown/knockout samples

• Immunoprecipitation followed by mass spectrometry:

  • Perform immunoprecipitation from samples of each species

  • Confirm pulled-down protein identity by mass spectrometry

  • Verify that the same protein is being detected across species

• Cross-validation with multiple antibodies:

  • Use different antibodies targeting distinct PCSK2 epitopes

  • Compare detection patterns across species

  • Consistent results with multiple antibodies increase confidence in specificity

• Immunohistochemistry comparison:

  • Compare staining patterns in homologous tissues across species

  • Evaluate subcellular localization consistency

  • Assess staining in tissues known to express or lack PCSK2 in each species

• Control experiments:

  • Include isotype controls for each species

  • Use secondary antibody-only controls

  • Test potential cross-reactivity with related proteins (other proprotein convertases)

This comprehensive validation strategy ensures reliable cross-species comparisons, as exemplified by studies that successfully detected PCSK2 in both human cell lines (MCF-7, HepG2) and mouse cell lines (beta TC-6) .