PNLIPRP2 Antibody, FITC conjugated

What is PNLIPRP2 and what cellular functions does it mediate?

PNLIPRP2 (Pancreatic lipase-related protein 2, also known as PL-RP2 or PLRP2) is a phospholipase A1 (PLA1) enzyme that plays crucial roles in membrane organization and protein localization. It functions as a key organizer of membrane protein localization, particularly at neurite tips in neuronal cells. PNLIPRP2 catalyzes the production of 1-oleoyl-2-palmitoyl-PC (OPPC) through acyl-chain remodeling of membrane phospholipids . This enzymatic activity creates specific lipid domains that facilitate the selective assembly of proteins like syntaxin 4 (Stx4). In turn, these organized membrane domains promote the fusion of transport vesicles containing proteins such as dopamine transporters, establishing functional membrane compartmentalization essential for neuronal function .

What are the optimal storage conditions for PNLIPRP2 Antibody, FITC conjugated?

For optimal preservation of PNLIPRP2 Antibody, FITC conjugated, store at -20°C or -80°C immediately upon receipt. The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . It is crucial to avoid repeated freeze-thaw cycles as these can compromise antibody activity and increase background fluorescence. For experiments requiring frequent use, consider aliquoting the antibody into smaller volumes before freezing to minimize freeze-thaw cycles .

What species reactivity has been confirmed for PNLIPRP2 Antibody?

The commercially available PNLIPRP2 Antibody, FITC conjugated has been specifically validated for human sample reactivity . Some antibodies against PNLIPRP2 (non-FITC conjugated) have been tested and confirmed to work with mouse and rat samples as well . When planning experiments with non-human samples, it's advisable to perform preliminary validation tests or consult with manufacturers about cross-reactivity potential. The antibody's target, recombinant Human Pancreatic lipase-related protein 2 protein (amino acids 305-460), shares varying degrees of homology with orthologs in other species, which may affect cross-reactivity .

How should PNLIPRP2 Antibody be used for immunofluorescence studies of neuronal membrane domains?

For immunofluorescence studies investigating PNLIPRP2 localization in neuronal membrane domains:

• Fix cells using 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour

• Apply PNLIPRP2 Antibody, FITC conjugated at a 1:100-1:500 dilution (optimize for your specific application)

• Incubate overnight at 4°C in a humidified chamber

• Wash extensively with PBS (3 × 5 minutes)

• Mount using anti-fade mounting medium

For co-localization studies with membrane proteins like syntaxin 4 (Stx4) or dopamine transporters, use appropriate complementary fluorophores that don't overlap with FITC's emission spectrum. Based on published research, PLRP2 has been observed to colocalize with OPPC during neurite extension in NGF-stimulated PC12 cells . When imaging, focus particularly on neurite tips where PLRP2 accumulates following NGF stimulation .

How can PNLIPRP2 Antibody be used for Western blotting applications?

For Western blotting detection of PNLIPRP2:

• Prepare cell lysates (15-20 μg protein per lane is recommended)

• Separate proteins using SDS-PAGE on 7.5-10% gels

• Transfer to PVDF membranes

• Block with 5% milk in TBST (blocking buffer)

• For unconjugated PNLIPRP2 antibodies: Dilute 1:2000-1:10000 in blocking buffer

• For FITC-conjugated antibodies: An HRP-conjugated anti-FITC secondary antibody will be needed

• Incubate overnight at 4°C

• Wash with TBST (3 × 10 minutes)

• Apply appropriate secondary antibody (if using unconjugated primary)

• Develop using HRP substrate and detect using X-ray film or CCD camera

The expected molecular weight for PNLIPRP2 is approximately 52 kDa . When analyzing results, note that PNLIPRP2 expression is inducible by factors such as NGF stimulation in PC12 cells, with a reported 6.5-fold increase in mRNA levels following NGF treatment .

What is the recommended protocol for using PNLIPRP2 Antibody in vesicle immunoprecipitation studies?

For vesicle immunoprecipitation involving PNLIPRP2:

• Homogenize neurons or neural cells in lysis buffer (typically containing protease inhibitors) using Potter homogenizers at 4°C

• Centrifuge homogenates at 1,000 × g to remove nuclei and cell debris

• Pre-clear the supernatant with control IgG and Protein G agarose

• Incubate with PNLIPRP2 Antibody (5-10 μg) overnight at 4°C

• Add Protein G agarose beads and incubate for 2 hours

• Wash extensively with lysis buffer

• Elute bound vesicles using gentle elution conditions

• Analyze vesicle contents through Western blotting or mass spectrometry

This approach has been adapted from previous studies investigating vesicular transport mechanisms . PNLIPRP2 has been implicated in controlling the localization of transport vesicles containing membrane proteins like dopamine transporters, making vesicle immunoprecipitation particularly valuable for studying these pathways.

How can CRISPR/Cas9-mediated knockout of PNLIPRP2 be verified using the FITC-conjugated antibody?

To verify CRISPR/Cas9-mediated knockout of PNLIPRP2:

• Design and implement CRISPR/Cas9 targeting of the PNLIPRP2 (PNLIPRP2) gene locus

• Isolate clonal strains following CRISPR editing

• Perform genomic verification through sequencing to confirm frameshift mutations

• Validate protein knockout through:

  • Western blotting using antibody against PNLIPRP2

  • Immunostaining using PNLIPRP2 Antibody, FITC conjugated

  • Functional assays for PLA1 activity using fluorogenic substrates like PED-A1

A successful knockout should show:

• Absence of PNLIPRP2 protein by Western blotting

• No detectable fluorescence signal in immunostaining

• Significantly reduced PLA1 activity in functional assays

This approach has been successfully employed in PC12 cells, where CRISPR/Cas9-mediated knockout of PNLIPRP2 abolished PLA1 activity at neurite tips and eliminated OPPC localization . For rescue experiments, consider introducing wild-type PNLIPRP2 with a fluorescent tag like DsRed to distinguish from endogenous protein .

How can PNLIPRP2 Antibody, FITC conjugated be used to investigate the relationship between lipid remodeling and membrane protein localization?

To investigate PNLIPRP2's role in lipid remodeling and protein localization:

• Use PNLIPRP2 Antibody, FITC conjugated to track PNLIPRP2 localization in cells

• Combine with lipid-specific probes or antibodies (like mAb#15 for OPPC detection)

• Track membrane protein distribution (e.g., syntaxin 4, dopamine transporters) using complementary fluorophores

• Perform time-course imaging following stimulation (e.g., NGF treatment in PC12 cells)

• Compare wild-type cells with PNLIPRP2 knockout cells

• Conduct rescue experiments with:

  • Wild-type PNLIPRP2

  • Catalytically inactive PNLIPRP2 (S184G, H295L mutations)

This multi-parameter approach allows for correlation between PNLIPRP2 enzymatic activity, lipid domain formation, and subsequent protein localization. Research has shown that PNLIPRP2 produces OPPC at neurite tips, which then assembles syntaxin 4 proteins within itself, facilitating the fusion of transport vesicles containing dopamine transporters .

What strategies can address potential cross-reactivity issues when using PNLIPRP2 Antibody in complex tissue samples?

To mitigate cross-reactivity concerns in complex tissue samples:

• Perform careful antibody validation:

  • Use PNLIPRP2 knockout tissues/cells as negative controls

  • Include absorption controls with recombinant PNLIPRP2 protein

  • Compare staining patterns with multiple antibodies targeting different PNLIPRP2 epitopes

• Optimize staining protocols:

  • Adjust antibody concentration (typically start with 1:100-1:400 dilution for IHC)

  • Modify blocking conditions (consider 5% normal serum from the same species as secondary antibody)

  • Test different antigen retrieval methods (if using fixed tissues, TE buffer pH 9.0 or citrate buffer pH 6.0)

• Employ complementary detection methods:

  • Combine immunostaining with in situ hybridization for PNLIPRP2 mRNA

  • Verify localization with biochemical fractionation and Western blotting

  • Use proximity ligation assays to confirm interactions with known binding partners

These approaches help ensure that observed signals truly represent PNLIPRP2 localization rather than non-specific binding or cross-reactivity with related proteins.

What are the common causes of high background when using FITC-conjugated antibodies, and how can they be addressed?

High background with FITC-conjugated PNLIPRP2 Antibody can result from several factors:

For particularly challenging samples, consider alternative detection methods such as using unconjugated primary PNLIPRP2 antibodies followed by highly cross-adsorbed secondary antibodies, or switching to longer-wavelength fluorophores that may have less autofluorescence issues.

How can researchers distinguish between active and inactive forms of PNLIPRP2 using antibody-based approaches?

Distinguishing active versus inactive PNLIPRP2 requires combining immunodetection with functional assays:

• Immunodetection strategies:

  • Use conformation-specific antibodies if available

  • Combine PNLIPRP2 Antibody, FITC conjugated with activity-based protein profiling probes

  • Apply proximity ligation assays to detect PNLIPRP2 interaction with known activators/inhibitors

• Functional validation approaches:

  • PLA1 activity assay using fluorogenic substrates like PED-A1

  • Monitor OPPC production using mAb#15 antibody

  • Compare wild-type cells with those expressing catalytically inactive PNLIPRP2 mutants

• Combined approaches:

  • Correlation analysis between PNLIPRP2 immunostaining intensity and PLA1 activity

  • Time-course studies following stimulation (e.g., NGF treatment)

  • Pharmacological modulation with lipase inhibitors

Research has demonstrated that mutations in the catalytic core of PNLIPRP2 (S184G and H295L) abolish its enzymatic activity while maintaining normal protein expression and localization . These mutants can serve as valuable controls to distinguish between protein presence and functional activity.

What considerations should be made when using PNLIPRP2 Antibody in primary neuronal cultures versus established cell lines?

When transitioning from established cell lines to primary neurons:

When working with primary neurons, it's crucial to consider that PNLIPRP2 expression and localization may vary with neuronal subtype, developmental stage, and activity state. Preliminary studies to characterize baseline PNLIPRP2 expression in your specific neuronal population are recommended before proceeding with detailed localization or functional studies.

How can PNLIPRP2 Antibody be used to investigate the role of lipid remodeling in neurological disorders?

PNLIPRP2 Antibody, FITC conjugated can facilitate investigations into lipid remodeling in neurological disorders through:

• Comparative analyses of PNLIPRP2 expression and localization in:

  • Post-mortem brain tissue from patients with neurological disorders

  • Animal models of neurodegenerative diseases

  • iPSC-derived neurons carrying disease-associated mutations

• Functional studies correlating PNLIPRP2 activity with:

  • Synapse formation and maintenance

  • Neurite outgrowth and pathfinding

  • Axonal transport disruptions

  • Membrane protein mislocalization

• Mechanistic investigations examining:

  • How disease-associated lipid alterations affect PNLIPRP2 function

  • Whether PNLIPRP2 dysfunction contributes to pathological protein aggregation

  • Potential therapeutic approaches targeting phospholipid remodeling pathways

Given PNLIPRP2's role in organizing membrane domains critical for protein localization , its dysfunction could contribute to membrane-related pathologies in various neurological conditions. The enzyme's involvement in creating specific lipid environments that facilitate protein assembly and vesicle fusion makes it a potential contributor to synaptic dysfunctions underlying many neurological disorders.

What methodological approaches can combine PNLIPRP2 immunodetection with lipidomic analyses?

Integrating PNLIPRP2 immunodetection with lipidomics requires specialized approaches:

• Spatial lipidomics with immunofluorescence:

  • Perform PNLIPRP2 immunostaining using FITC-conjugated antibody

  • Mark regions of interest based on PNLIPRP2 localization

  • Apply laser capture microdissection to isolate specific cellular regions

  • Conduct targeted lipidomic analysis on microdissected samples

  • Correlate lipid profiles with PNLIPRP2 abundance

• Cell sorting-based approaches:

  • Use PNLIPRP2 Antibody, FITC conjugated for fluorescence-activated cell sorting

  • Isolate PNLIPRP2-high versus PNLIPRP2-low cell populations

  • Perform comparative lipidomic analysis between populations

  • Identify lipid species enriched in PNLIPRP2-high cells

• Temporal correlation studies:

  • Track PNLIPRP2 expression/localization during stimulation (e.g., NGF treatment)

  • Collect parallel samples for lipidomic analysis at defined timepoints

  • Correlate changes in PNLIPRP2 activity with lipid composition alterations

  • Focus particularly on 1-oleoyl-2-palmitoyl-PC (OPPC) levels, which have been directly linked to PNLIPRP2 activity

These approaches can provide critical insights into how PNLIPRP2's phospholipase activity specifically remodels the membrane lipidome to create functional domains for protein localization.