PCSK2 Antibody, FITC conjugated

What is PCSK2 and what cellular functions does it serve?

PCSK2 (Proprotein Convertase Subtilisin/Kexin Type 2) is a serine endopeptidase involved in processing hormone and protein precursors at sites containing pairs of basic amino acid residues. It functions as a major proteolytic processing enzyme in the regulated secretory pathway of the neuroendocrine system, where it generates numerous hormones and neuropeptides. Most notably, PCSK2 is responsible for the release of glucagon from proglucagon in pancreatic A cells .

The enzyme undergoes autoactivation in post-Golgi compartments of the secretory system. Critically, interaction with the secretory protein 7B2 is required for proper activation of PCSK2 - the N-terminal domain of 7B2 stabilizes active PCSK2, while a C-terminal fragment can inhibit its activity . This regulatory mechanism ensures precise control over PCSK2 activity in neuroendocrine cells.

Which tissue types show optimal PCSK2 expression for positive controls?

Based on immunohistochemical studies, researchers should consider the following tissues as positive controls for PCSK2 antibody validation:

Human adrenal gland sections are particularly valuable as they provide internal negative controls (adrenal cortex) alongside positive staining in the medulla, as demonstrated in multiplexed immunofluorescence studies . For cell culture work, TT cells (human thyroid carcinoma epithelial cells) show reliable PCSK2 expression .

What are the optimal fixation and permeabilization protocols for PCSK2 immunofluorescence?

For reliable FITC-conjugated PCSK2 antibody detection, optimize your protocols based on sample type:

For cultured cells:

• Fix with 4% paraformaldehyde in PBS for 30 minutes at room temperature

• For intracellular staining, permeabilize with 90% methanol (as validated for TT cells)

• Alternatively, use 0.1% Triton X-100 for 10 minutes for milder permeabilization

• Block with 5% normal serum (matching secondary antibody species) in PBS with 0.1% Tween-20

For tissue sections:

• For FFPE sections, perform heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes

• This specific retrieval protocol has been validated for PCSK2 antibodies in multiplexed immunofluorescence

• For cryosections, fixation in cold acetone or 4% PFA provides adequate preservation of PCSK2 epitopes

The permeabilization step is critical since PCSK2 is primarily localized to the secretory pathway, requiring sufficient membrane permeabilization for antibody access to intracellular compartments.

How can I validate the specificity of my PCSK2-FITC antibody?

A systematic validation approach ensures reliable results with PCSK2-FITC antibodies:

• Western blot verification: Confirm detection of the expected ~70-71 kDa band in positive control lysates (human thyroid carcinoma cells or brain tissue) . The pro-form of PC2 is observed at approximately 70 kDa, consistent with literature reports .

• Positive control tissues/cells:

  • Human adrenal medulla (shows distinct staining pattern)

  • Small intestine NETs (consistently PCSK2-positive)

  • TT cell line (validated for PCSK2 expression)

• Negative controls:

  • Isotype control antibodies (e.g., Rabbit monoclonal IgG)

  • Omission of primary antibody while maintaining all other steps

  • Include tissues known to lack PCSK2 expression as biological negative controls

• Flow cytometric validation: If using PCSK2-FITC for flow cytometry, compare staining intensity against isotype controls and FMO (Fluorescence Minus One) controls to establish positive signal thresholds .

Proper validation not only confirms antibody specificity but also establishes optimal working concentrations for your specific experimental system.

How do different processing states of PCSK2 affect antibody detection?

PCSK2 exists in multiple processing states that can affect antibody recognition:

• Pro-enzyme form (~70 kDa): The inactive precursor form contains an N-terminal propeptide that must be cleaved for activation. Some antibodies preferentially recognize epitopes in this region.

• Mature enzyme (~64-66 kDa): Following autocatalytic cleavage of the propeptide, conformational changes may expose or mask certain epitopes.

• Complex with 7B2: Interaction with the secretory protein 7B2 is essential for proper PCSK2 activation . This binding can potentially alter epitope accessibility.

For comprehensive detection, consider these strategies:

• Verify which form(s) your specific antibody clone recognizes using Western blot

• For recombinant PCSK2 expression, co-express 7B2 to facilitate proper activation

• When studying processing dynamics, use antibodies that can distinguish between pro-PCSK2 and mature PCSK2

• Be aware that processing efficiency varies between cell types, potentially affecting detection sensitivity

Understanding which form your antibody recognizes is crucial for correctly interpreting experimental results, especially when studying PCSK2 processing and activation dynamics.

What are the technical considerations for multiplexing PCSK2-FITC antibodies with other fluorophores?

Successful multiplexed immunofluorescence with PCSK2-FITC antibodies requires careful optimization:

• Spectral compatibility planning:

  • FITC (excitation ~495nm, emission ~520nm) can show spectral overlap with GFP and other green fluorophores

  • Pair with spectrally distinct fluorophores like DAPI (blue), Cy3/TRITC (red), and Cy5/APC (far-red)

  • The search results describe successful multiplexing using an Opal 4-color kit with PCSK2

• Signal balancing strategies:

  • Reserve FITC for moderately abundant targets like PCSK2 in positive tissues

  • Use brighter fluorophores (e.g., Alexa Fluor 647) for low-abundance targets

  • Consider tyramide signal amplification for FITC channel if needed (validated in published protocols)

• Cross-reactivity prevention:

  • Use primary antibodies from different host species

  • If using same-species antibodies, employ sequential staining with blocking steps

  • The search results describe successful sequential staining approaches for PCSK2 with other markers

• Successful multiplexed panels with PCSK2:

  • PCSK2 + C11B2/CYP11B2 + SULT2A1 (validated combination)

  • PCSK2 + CYP11A1 + Collagen VI (validated combination)

  • Include DAPI as nuclear counterstain in all panels

These considerations are essential for generating reliable multiplexed data that accurately represents the biological relationship between PCSK2 and other proteins of interest.

How can PCSK2 antibody staining patterns differentiate neuroendocrine tumor subtypes?

PCSK2 immunostaining has emerged as a valuable diagnostic marker for specific neuroendocrine tumor (NET) subtypes:

This distinct expression pattern makes PCSK2 particularly valuable for identifying the primary site of metastatic NETs when the origin is unknown . In research contexts, consider these applications:

• Include PCSK2 in diagnostic immunophenotyping panels for NET classification

• Use PCSK2 staining intensity to potentially correlate with functional status of different NET subtypes

• Combine PCSK2 with other neuroendocrine markers for comprehensive tumor characterization

• Employ FITC-conjugated PCSK2 antibodies in flow cytometry to quantitatively assess expression levels across tumor subtypes

The consistency of PCSK2 expression in midgut NETs makes it particularly valuable as a research tool for investigating the biology of these tumor types.

What factors affect variability in PCSK2 antibody staining intensity?

When encountering variability in PCSK2-FITC staining intensity, consider these biological and technical factors:

Biological variables:

• Processing state heterogeneity: Cells may contain varying ratios of pro-PCSK2 to mature PCSK2

• 7B2 co-expression levels: Since 7B2 is required for proper PCSK2 activation, variable 7B2 expression can affect PCSK2 processing efficiency

• Secretory pathway status: PCSK2 trafficking and localization vary with secretory activity

• Cell-type specific post-translational modifications: Different glycosylation patterns may affect epitope accessibility

Technical variables:

• Fixation effects: Overfixation can reduce signal by masking epitopes

• Antibody clone specificity: Different clones (e.g., 694009 vs. EPR23578-19 ) may have different affinities

• Antigen retrieval efficiency: Insufficient retrieval can limit antibody access to epitopes

• Detection system sensitivity: Direct FITC conjugates vs. amplification systems significantly impact signal intensity

To address variability:

• Standardize fixation and staining protocols across experiments

• Include reference standards with known PCSK2 expression levels

• Consider tyramide signal amplification for low-abundance applications

• When comparing expression levels, process all samples simultaneously with identical protocols

Understanding these factors allows for more accurate interpretation of PCSK2 staining patterns across different experimental systems.

What is the optimal protocol for PCSK2 immunoblotting?

For reliable Western blot detection of PCSK2, follow this optimized protocol based on validated research methods:

Sample preparation:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples under reducing conditions (as specified in successful protocols)

• Load 20-40 μg total protein per lane (40 μg of cell lysate was validated)

Electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels to properly resolve PCSK2 (~70 kDa)

• Transfer to PVDF membrane (validated for PCSK2 detection)

• Verify transfer efficiency with reversible protein stain

Immunodetection:

• Block with 5% non-fat dry milk in TBST (validated blocking condition)

• Incubate with primary PCSK2 antibody at 1:1000 dilution (validated working concentration)

• Wash thoroughly with TBST (minimum 3×10 minutes)

• Incubate with appropriate HRP-conjugated secondary antibody (1:50,000 dilution for anti-rabbit HRP was successful)

• Develop using ECL substrate with 30-40 second exposure time (37 seconds was optimal in published work)

Expected results:

• Primary band at approximately 70 kDa (pro-PCSK2)

• Potential secondary bands representing processing intermediates

• Validated positive controls include TT cells and human brain (cerebellum) tissue

This protocol has been validated for detecting both endogenous PCSK2 and recombinant tagged versions in various experimental systems.

How can flow cytometry protocols be optimized for PCSK2-FITC antibody applications?

Flow cytometry with PCSK2-FITC antibodies requires optimization for this primarily intracellular protein:

Cell preparation:

• Harvest cells using non-enzymatic methods when possible to preserve surface epitopes

• Fix with 4% paraformaldehyde for 15-30 minutes at room temperature

• Permeabilize with 90% methanol (validated for PCSK2 detection in TT cells)

• Block with 5% normal serum in permeabilization buffer for 30 minutes

Staining protocol:

• Incubate with PCSK2-FITC antibody at 1:500 dilution (0.1μg per test is a validated starting concentration)

• Incubate for 1 hour at room temperature or overnight at 4°C

• Wash twice with permeabilization buffer

• If cell autofluorescence is high, consider post-staining with an autofluorescence quencher

Critical controls:

• Unstained cells to establish autofluorescence baseline

• Isotype control-FITC to determine non-specific binding (validated using Rabbit IgG monoclonal isotype control)

• FMO (Fluorescence Minus One) controls for multicolor panels

• Positive control cells (TT cell line is validated for PCSK2 expression)

Analysis considerations:

• Set FITC voltage to properly visualize full distribution of positive populations

• Gate strategy should include:

  • FSC/SSC to exclude debris

  • FSC-H/FSC-A to select singlets

  • Live/dead discrimination if applicable

  • PCSK2-FITC signal analysis on defined cell populations

This protocol provides a starting point that should be further optimized for your specific cell type and experimental questions.

How can autofluorescence be minimized when using FITC-conjugated PCSK2 antibodies?

Autofluorescence can significantly impact FITC-conjugated antibody detection, particularly in tissues that naturally contain fluorescent compounds. Implement these strategies to maximize signal-to-noise ratio:

Pre-treatment approaches:

• Tissue pre-processing: Treat sections with 0.1-0.3% Sudan Black B in 70% ethanol for 10-20 minutes after antibody incubation

• Chemical quenching: Incubate samples with 1 mg/ml NaBH₄ in PBS for 10 minutes before antibody staining

• Photobleaching: Expose tissues to strong illumination in PBS prior to staining to reduce endogenous fluorophore contribution

Acquisition strategies:

• Spectral unmixing: Acquire autofluorescence signature from unstained regions and mathematically subtract from FITC signal

• Narrow bandpass filters: Use filters that precisely match FITC emission peak while excluding common autofluorescence wavelengths

• Time-gated detection: If using confocal microscopy with pulsed excitation, employ time-gated detection to separate FITC signal from shorter-lifetime autofluorescence

Alternative approaches:

• Signal amplification: Use tyramide signal amplification (TSA) systems, which have been successfully employed with PCSK2 antibodies

• Alternative detection methods: Consider other fluorophores or enzymatic detection systems if autofluorescence remains problematic

Tissue-specific considerations:

• Neuroendocrine tissues often contain lipofuscin, which emits broadly in yellow-orange spectrum

• Formalin-fixed tissues develop formaldehyde-induced fluorescence that overlaps with FITC

• Elastin and collagen have intrinsic blue-green fluorescence that may interfere with FITC detection

Implementing these strategies will significantly improve the specificity and sensitivity of PCSK2-FITC detection, particularly in challenging sample types like aged neuroendocrine tissues.

What are the best practices for multiplexed immunohistochemistry with PCSK2 antibodies?

Multiplexed immunohistochemistry (mIHC) with PCSK2 antibodies requires careful planning and optimization. Based on successful research applications, follow these guidelines:

Protocol optimization:

• Antibody validation: First validate PCSK2 antibody in single-color IHC before multiplexing

• Antibody dilution: Determine optimal dilution for PCSK2 antibodies in multiplex context (1:2000 dilution was successful in published research)

• Antigen retrieval: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes provides optimal epitope exposure

• Staining sequence: Determine optimal sequence for multiple antibodies (PCSK2 was successfully used as the first antibody in sequential staining protocols)

Validated multiplex panels:

• Adrenal gland characterization panel:

  • PCSK2 (adrenal medulla marker)

  • C11B2/CYP11B2 (zona glomerulosa marker)

  • SULT2A1 (zona reticularis marker)

  • DAPI (nuclear counterstain)

• Alternative adrenal tissue panel:

  • PCSK2 (adrenal medulla marker)

  • CYP11A1 (adrenal cortex marker)

  • Collagen VI (extracellular matrix marker)

  • DAPI (nuclear counterstain)

Technical implementation:

• Signal amplification: Employ tyramide signal amplification systems for each round of staining

• Heat-based antibody stripping: Between antibody rounds, use heat treatment to remove previous antibodies

• Image acquisition: Use confocal microscopy for optimal signal separation and colocalization analysis

• Automated platforms: Consider automated staining platforms (like BOND RX) for consistent results

These approaches have been successfully implemented on formalin-fixed paraffin-embedded tissues, demonstrating the compatibility of PCSK2 antibodies with complex multiplexed protocols.

How can PCSK2 antibodies be used to study protein-protein interactions?

Investigating PCSK2 interactions with partners like 7B2 and potential novel interactors requires specialized approaches:

Co-immunoprecipitation (Co-IP):

• Lysis conditions: Use non-denaturing lysis buffers to preserve protein-protein interactions

  • 25 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • Protease inhibitor cocktail

• Antibody selection: Choose PCSK2 antibodies validated for immunoprecipitation applications

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg antibody per mg of total protein

• Controls:

  • Input (5-10% of pre-cleared lysate)

  • IgG control (non-specific antibody of same isotype)

  • Reverse IP (immunoprecipitate with antibody against suspected interaction partner)

Proximity Ligation Assay (PLA):
This technique can detect protein interactions with high sensitivity and spatial resolution:

• Primary antibodies against PCSK2 and potential partner protein (from different species)

• Species-specific secondary antibodies with attached oligonucleotides

• When proteins are in close proximity (<40 nm), oligonucleotides can be ligated and amplified

• Detection of amplified DNA indicates protein proximity/interaction

FRET (Förster Resonance Energy Transfer):
For studying interactions in living cells:

• Express PCSK2 tagged with donor fluorophore

• Express potential interaction partner tagged with acceptor fluorophore

• Energy transfer between fluorophores indicates protein proximity

• Can be analyzed by fluorescence microscopy or flow cytometry

Considerations specific to PCSK2:

• 7B2 interaction is well-established and can serve as positive control

• PCSK2 primarily localizes to the secretory pathway, so interaction studies should focus on this compartment

• The TAZ/β-Trcp degradation pathway may be relevant for understanding PCSK2 regulation

How does PCSK2 expression correlate with functional activity in neuroendocrine systems?

The relationship between PCSK2 expression and functional activity in neuroendocrine systems involves complex regulatory mechanisms:

Processing efficiency:

• PCSK2 requires 7B2 for proper activation

• Expression levels of both PCSK2 and 7B2 determine processing capacity

• The ratio between pro-PCSK2 and mature PCSK2 may better indicate functional status than total PCSK2

Substrate availability:

• PCSK2 processes multiple hormone precursors including proglucagon, proinsulin, and proopiomelanocortin

• Co-expression of PCSK2 with its substrates is necessary for functional relevance

• Different cell types may contain different PCSK2 substrates, affecting functional outcomes

Research approaches:

• Activity assays: Measure PCSK2 enzymatic activity using fluorogenic substrates in parallel with expression analysis

• Substrate processing: Quantify ratios of precursor to mature hormones (e.g., proglucagon to glucagon)

• Cell-specific analysis: Use multiplexed immunofluorescence to correlate PCSK2 expression with hormone content in specific cell types

Pathological correlations:

• In NETs, PCSK2 expression patterns correlate with tumor subtype and origin

• Altered PCSK2 expression has been implicated in diabetes-related pathways and Alzheimer's disease

• Functional consequences of aberrant PCSK2 expression may include dysregulated hormone processing

These complex relationships require integrated analysis of expression, localization, processing state, and functional outcomes to fully understand PCSK2's role in health and disease.

What role does PCSK2 play in neurodegenerative disease pathways?

Emerging research suggests connections between PCSK2 and neurodegenerative processes:

Alzheimer's Disease connections:

• The search results reference a study examining "Altered Expression of Diabetes-Related Genes in Alzheimer's Disease Brains: The Hisayama Study"

• This suggests potential links between PCSK2's role in hormone processing and AD pathophysiology

• PCSK2 is expressed in various brain regions, including the cerebellum

Mechanistic possibilities:

• Neuropeptide processing: PCSK2 processes numerous neuropeptides crucial for neuronal health and function

• Insulin/insulin-like growth factor processing: Disruption may affect insulin signaling in the brain, which is increasingly recognized in neurodegeneration

• Inflammatory mediator regulation: Altered processing of peptides involved in neuroinflammation

Experimental approaches:

• Immunohistochemical analysis of PCSK2 in neurodegenerative disease brain samples

• Correlation of PCSK2 expression with markers of neurodegeneration

• Functional studies in neuronal models with manipulated PCSK2 expression

Potential therapeutic implications:

• PCSK2 could represent a novel target for addressing specific aspects of neurodegenerative diseases

• Modulating PCSK2 activity might normalize processing of neuroprotective peptides

This remains an emerging area where PCSK2 antibodies serve as critical tools for exploring these potential connections between neuroendocrine function and neurodegeneration.

How do post-translational modifications affect PCSK2 function and antibody detection?

PCSK2 undergoes several post-translational modifications that influence both its function and detection by antibodies:

Key modifications:

• Proteolytic processing:

  • Conversion from ~70 kDa pro-PCSK2 to mature ~64 kDa enzyme

  • Requires autocatalytic cleavage of N-terminal propeptide

  • Antibodies targeting different regions may preferentially detect specific forms

• Glycosylation:

  • N-linked glycosylation affects PCSK2 folding and stability

  • Can alter antibody accessibility to certain epitopes

  • May create heterogeneity in apparent molecular weight on Western blots

• Phosphorylation:

  • Potential regulatory mechanism affecting PCSK2 activity

  • May create conformational changes affecting antibody binding

Impact on antibody detection:

Experimental considerations:

• When studying processing dynamics, use antibodies targeting both pro-region and mature region

• For quantitative analysis, understand which form(s) your antibody recognizes

• Consider sample preparation methods that preserve or remove specific modifications depending on experimental questions

Understanding these modifications is critical for correctly interpreting antibody-based detection results and their biological significance.

What are emerging applications for PCSK2 antibodies in diabetes research?

PCSK2 plays critical roles in glucose homeostasis through its processing of prohormones, making PCSK2 antibodies valuable tools in diabetes research:

Key research applications:

• Pancreatic islet biology:

  • Studying α-cell function through PCSK2-mediated glucagon processing

  • Investigating paracrine interactions between islet cell types

  • Examining PCSK2 expression changes during islet development or in disease states

• Enteroendocrine system:

  • Research on GLP-1 secretion from intestinal L-cells (referenced in a study using Costus pictus D. Don leaf extract)

  • Investigation of gut-derived hormones in glucose regulation

• Diabetes complications:

  • Examining links between altered PCSK2 expression and diabetes-related changes in neural tissues

  • Potential connections to diabetes-related cognitive decline

Methodological approaches:

• Multiplex immunofluorescence:

  • Combine PCSK2-FITC with markers of different islet cell types

  • Correlate PCSK2 expression with functional markers of islet stress

  • Study co-localization with insulin processing machinery

• Flow cytometry:

  • Quantify PCSK2 expression in sorted pancreatic cell populations

  • Measure changes in PCSK2 levels in response to metabolic challenges

• In vitro functional studies:

  • Assess impact of PCSK2 modulation on hormone processing and secretion

  • Screen compounds for effects on PCSK2 expression or activity

These applications highlight the importance of PCSK2 antibodies in understanding the complex interplay between neuroendocrine function and metabolic regulation in both health and diabetes.

How can PCSK2 antibodies be used to study protein degradation pathways?

PCSK2 antibodies can provide insights into protein degradation mechanisms, particularly in the context of regulated proteolysis:

Protein degradation pathways relevant to PCSK2:

• Ubiquitin-proteasome system:

  • The search results mention a connection between polycystin-2 (PC2) and the SCFβ-Trcp E3 ligase complex

  • While this specifically refers to PC2 (not PCSK2), similar pathways may regulate PCSK2 turnover

  • PCSK2 antibodies can help investigate potential ubiquitination and proteasomal degradation

• Lysosomal degradation:

  • As a secretory pathway protein, PCSK2 may undergo lysosomal degradation

  • Antibodies can track PCSK2 trafficking to lysosomes under various conditions

• Regulated intramembrane proteolysis:

  • PCSK2 processing may involve membrane-associated proteolytic events

  • Antibodies recognizing different domains can help map processing sites

Experimental approaches:

• Pulse-chase analysis:

  • Metabolically label newly synthesized proteins

  • Immunoprecipitate PCSK2 at various chase times

  • Quantify degradation rates under different conditions

• Pharmacological interventions:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Apply lysosomal inhibitors (e.g., chloroquine, bafilomycin A1)

  • Use PCSK2 antibodies to detect changes in steady-state levels or localization

• Co-immunoprecipitation studies:

  • Identify potential interactions with degradation machinery components

  • Investigate whether PCSK2 associates with E3 ubiquitin ligases

  • Study potential interactions with SCFβ-Trcp components

• In vitro degradation assays:

  • Similar to the ubiquitination assay described for PC2

  • Use purified components to reconstitute potential degradation pathways

  • Detect PCSK2 degradation products with domain-specific antibodies

These approaches can reveal mechanisms controlling PCSK2 turnover, adding another layer to our understanding of its regulation in neuroendocrine systems.