SYNJ1 Antibody, FITC conjugated

What is Synaptojanin-1 (SYNJ1) and why is it significant in neuroscience research?

Synaptojanin-1 (SYNJ1) is a brain-enriched phosphoinositide phosphatase that plays critical roles in synaptic vesicle recycling, endocytosis, and phosphoinositide metabolism. It contains two phosphatase domains: the Sac1 domain that dephosphorylates PI4P, PI3P, and PI(3,5)P2, and a 5-phosphatase domain that primarily acts on PI(4,5)P2 and PI(3,4,5)P3 .

SYNJ1 is significant in neuroscience research because:

• It is essential for normal synaptic transmission and vesicle trafficking

• The SYNJ1 gene maps to chromosome 21, making it relevant for Down syndrome studies

• Mutations in SYNJ1 have been linked to early-onset Parkinson's disease and other neurological disorders

• It shows altered expression and localization in Alzheimer's disease, particularly in relation to APOE genotype

For maximum stability and performance of SYNJ1 Antibody, FITC conjugated:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, which can reduce activity and increase background fluorescence

• The antibody is typically provided in a storage buffer containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as preservative

• For working solutions, store at 4°C and use within 1-2 weeks

• Protect from light due to the photosensitivity of the FITC fluorophore

How can researchers optimize immunofluorescence protocols for SYNJ1 detection in brain tissue sections?

Optimizing immunofluorescence for SYNJ1 detection in brain tissue requires careful consideration of several factors:

Sample preparation:

• For human post-mortem brain tissue, use 10-micron cryosections from perfusion-fixed tissue

• For optimal morphological preservation and antigen accessibility, consider using 4% paraformaldehyde fixation with controlled post-fixation time (12-24 hours)

Antigen retrieval:

• Perform heat-mediated antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• For enhanced detection in brain tissue, particularly in regions with high lipid content, a mild detergent permeabilization step (0.1-0.3% Triton X-100) is recommended

Signal amplification:

• For weakly expressed SYNJ1, consider using Tyramide Signal Amplification (TSA) systems:

  • Example: Tyramide-FITC kit with biotinylated secondary antibody as demonstrated in studies of SYNJ1 in AD brain tissue

• Double immunofluorescence protocols can be employed to co-localize SYNJ1 with markers like synaptophysin, PHF-tau, or actin

Imaging considerations:

• Use confocal microscopy for detailed synaptic localization (Olympus Fluoview Fv1000 or similar)

• Employ ApoTome or similar structured illumination systems for improved signal-to-noise ratio

• Control for autofluorescence, which is common in aged brain tissue, by using appropriate emission filters and sequential scanning

What are the critical considerations when designing experiments to study SYNJ1 in neurodegenerative disease models?

When investigating SYNJ1 in neurodegenerative disease models, researchers should consider:

Genetic background influences:

• APOE genotype significantly affects SYNJ1 expression and localization in Alzheimer's disease, with higher levels observed in APOE ε4 carriers

• Control for genetic background in mouse models, especially when using models with altered APOE status

Cell-type specific expression:

• SYNJ1 is predominantly expressed in neurons but also present in astrocytes, microglia, and oligodendrocytes at lower levels

• Consider cell-type specific analyses for accurate interpretation of results:

Disease-specific protein interactions:

• In Alzheimer's disease: Examine co-localization with amyloid plaques and dystrophic neurites

• In Parkinson's disease: Investigate interaction with auxilin and relationship with parkin levels

• In SCI models: Assess SYNJ1 in relation to functional recovery and PIP2 levels

Solubility fractionation:

• SYNJ1 shows altered solubility in AD brains, with increased presence in sarkosyl-insoluble fractions containing PHF-tau

• Consider sequential extraction protocols to separate different protein pools (RIPA-soluble vs. insoluble)

How can researchers accurately interpret changes in SYNJ1 localization or expression in disease conditions?

Accurate interpretation of SYNJ1 alterations requires:

Quantitative approaches:

• Use standardized thresholding analyses in ImageJ for immunofluorescence quantification

• For transcript analysis, normalize SYNJ1 expression to multiple reference genes such as Peptidylprolyl Isomerase B (pPib), Ring Finger Protein 4 (RNF4), and DNA-directed RNA polymerase II subunit RPB1 (PolR2A)

Disease-specific patterns:

• In Alzheimer's disease:

  • SYNJ1 accumulates in Hirano bodies (actin-positive structures)

  • SYNJ1 localizes to plaque-associated dystrophic neurites

  • Some neurofibrillary tangles show SYNJ1 immunoreactivity

  • Higher immunoreactivity in neurons and senile plaques of APOE ε4 carriers

Post-translational modifications:

• Consider potential cleavage of SYNJ1 by calpain, which is highly activated in AD brains

• Western blot analysis may reveal both full-length SYNJ1 and cleaved fragments

• In disease states, SYNJ1 may appear as a smear rather than discrete bands in insoluble fractions

Contradictory findings resolution:

• Apparent differences in SYNJ1 levels between transcript and protein analyses may reflect altered protein stability or solubility rather than transcriptional regulation

• Different antibodies may yield varying results depending on which epitope they recognize and whether that region is affected by post-translational modifications

What methodological approaches are recommended for studying the relationship between SYNJ1 dysfunction and phosphoinositide metabolism?

To investigate SYNJ1's role in phosphoinositide metabolism:

Structural and functional analysis:

• The catalytic 5-phosphatase domain of human SYNJ1 has been crystallized, providing insights into substrate binding and catalytic mechanism

• Nanobody-aided crystallography has been successful in resolving SYNJ1 structure with trapped substrate

Mutation impact assessment:

• For analyzing clinical mutations (like Y793C, R800C, Y849C), assess their effect on catalytic activity through in vitro phosphatase assays

• Molecular dynamics simulations can complement experimental approaches to understand structural effects of mutations

Phosphoinositide visualization:

• PIP2 levels can be assessed using specific antibodies (e.g., Echelon Z-P034 antibody)

• For dynamic studies, consider fluorescent phosphoinositide sensors or FRET-based approaches

Rescue experiments:

• In knockout models, compare rescue efficacy between wild-type SYNJ1 and phosphatase-defective mutants

• Different phenotypes may depend on specific phosphatase domains (Sac domain vs. 5-phosphatase domain)

How can researchers troubleshoot common technical issues with SYNJ1 Antibody, FITC conjugated in immunofluorescence applications?

For advanced dual-labeling experiments:

• Ensure antibodies are raised in different host species

• If using antibodies from the same species, employ sequential staining with complete blocking between steps

• Consider spectral overlap when choosing fluorophore combinations

What are the key considerations when designing experiments to study SYNJ1's role in synaptic vesicle recycling?

When investigating SYNJ1's function in synaptic vesicle recycling:

Model systems selection:

• Primary neuronal cultures (cortical or hippocampal) are valuable for detailed imaging of synaptic processes

• Cultured neurons from SYNJ1 mutant models (such as R258Q Parkinsonism mutation) show distinctive clustering of endocytic proteins at synapses

Experimental approaches:

• Clathrin-mediated endocytosis assessment:

  • Immunofluorescence for clathrin, AP2, auxilin, endophilin, and amphiphysin can reveal endocytic protein clustering

  • Electron microscopy provides direct visualization of coated vesicle accumulation

  • pHluorin-based assays can assess rates of endocytosis and recycling

Genetic manipulation strategies:

• For rescue experiments in SYNJ1-deficient neurons, consider testing:

  • Wild-type SYNJ1

  • Sac phosphatase-defective mutants

  • 5-phosphatase defective mutants

  • This approach can distinguish the contributions of each phosphatase domain

Temporal considerations:

• SYNJ1 phenotypes in cultured neurons can be observed as early as DIV7 but become more prominent by DIV19

• For in vivo studies, consider both developmental and maintenance roles of SYNJ1

How do you integrate SYNJ1 protein analysis with functional studies in neurological disorder research?

Effective integration of SYNJ1 protein analysis with functional studies requires:

Multi-level analysis approach:

• Molecular level: Assess SYNJ1 expression, localization, post-translational modifications

• Cellular level: Examine effects on endocytosis, phosphoinositide metabolism, autophagy

• Circuit level: Evaluate synaptic transmission, plasticity

• Behavioral level: Measure relevant phenotypes (motor, cognitive)

Disease-specific functional readouts:

• For Parkinson's disease models:

  • Measure dopaminergic neuron survival

  • Assess motor function (rotarod, open field)

  • Analyze α-synuclein pathology

• For Alzheimer's disease models:

  • Evaluate amyloid and tau pathology

  • Test memory performance

  • Analyze synaptic density and function

Translational biomarker development:

• Consider how SYNJ1 alterations could serve as biomarkers

• Correlate SYNJ1 protein levels or modifications with disease progression

• Assess whether SYNJ1 changes precede clinical symptoms

Experimental timeline planning:

• For genetic models, implement longitudinal designs spanning:

  • Presymptomatic stages

  • Disease onset

  • Progressive phases

• This approach allows correlation of SYNJ1 changes with disease progression

What are the essential controls needed when using SYNJ1 Antibody, FITC conjugated for quantitative research?

For rigorous quantitative research using SYNJ1 Antibody, FITC conjugated:

Antibody validation controls:

• Positive control: Brain tissue/cells with known SYNJ1 expression

• Negative control: SYNJ1 knockout tissue/cells or siRNA-treated samples

• Peptide competition assay: Pre-absorption with immunizing peptide should abolish specific signal

• Isotype control: Use irrelevant FITC-conjugated antibody of same isotype to assess non-specific binding

Quantification controls:

• Internal normalization standards: Co-stain with markers of specific subcellular compartments

• Technical replicates: Minimum of three independent staining experiments

• For fluorescence quantification:

  • Include calibration standards

  • Control for photobleaching

  • Use identical acquisition parameters across all samples

Disease model-specific controls:

• Age-matched controls when studying neurodegenerative conditions

• APOE genotype-matched controls for AD studies

• For human tissue studies, control for post-mortem interval and fixation conditions

Statistical validation:

• Power analysis to determine appropriate sample size

• Blinded quantification to prevent bias

• Appropriate statistical tests based on data distribution

How can SYNJ1 Antibody, FITC conjugated be used to study disease mechanisms in Alzheimer's and Parkinson's disease models?

SYNJ1 Antibody, FITC conjugated offers valuable insights into disease mechanisms:

Alzheimer's disease applications:

• Co-localization studies: SYNJ1 accumulates in specific AD pathological structures:

  • Dystrophic neurites surrounding amyloid plaques

  • Hirano bodies (actin-positive structures)

  • Some neurofibrillary tangles

• APOE interaction: SYNJ1 levels are higher in neurons and senile plaques of AD patients with APOE ε4 allele(s)

  • Dual labeling with APOE and SYNJ1 can reveal genotype-specific patterns

• Protein solubility studies: SYNJ1 co-enriches with PHF-tau in sarkosyl-insoluble fractions of AD brain

Parkinson's disease applications:

• Mutation effect visualization: Compare localization of wild-type vs. mutant SYNJ1 (e.g., R258Q Parkinsonism mutation)

• Endocytic protein clustering: SYNJ1 mutations lead to distinctive synaptic clustering of endocytic proteins that can be visualized with immunofluorescence

• Parkin interaction: SYNJ1 mutations affect levels of the E3 ubiquitin ligase parkin (PARK2), potentially connecting different genetic forms of Parkinson's disease

Technical approach:

• For optimal visualization of SYNJ1 in disease models, use confocal microscopy with high NA objectives

• Z-stack acquisition followed by maximum intensity projection or 3D reconstruction provides comprehensive spatial information

• Quantitative co-localization analysis can be performed using Manders or Pearson coefficients

What methodological approaches are recommended for analyzing SYNJ1 in spinal cord injury models?

For spinal cord injury (SCI) research involving SYNJ1:

Tissue preparation:

• Use perfusion-fixed spinal cord tissue sectioned at 10 microns for optimal immunofluorescence

• For SYNJ1 detection in spinal cord, antibodies such as Novus NBP1-87842 have been successfully employed

Regional analysis considerations:

• SYNJ1 is predominantly expressed in the gray matter of spinal cord sections

• Cell-type specific expression analysis shows:

  • Highest expression in neurons

  • Lower levels in astrocytes, microglia, and oligodendrocytes

Functional correlation:

• SYNJ1 levels correlate with locomotor function recovery after SCI in ApoE4 mouse models

• Genetic reduction of SYNJ1 improves locomotor function recovery at 14 days post-SCI in ApoE4 mice

Experimental design recommendations:

• Include time-course analysis (SYNJ1 expression remains relatively constant after SCI)

• Compare gray matter vs. white matter SYNJ1 expression

• Analyze sections both above and at the lesion site

• Consider APOE genotype as a variable affecting SYNJ1 expression after injury

How can single-cell techniques be integrated with SYNJ1 immunofluorescence for advanced neuroscience research?

Integrating single-cell approaches with SYNJ1 immunofluorescence offers powerful insights:

Single-cell RNA-seq correlation:

• Mining single-nucleus RNA-seq datasets (such as spinal cord cell atlases) can identify cell types with highest SYNJ1 expression

• Immunofluorescence can then validate protein-level expression in identified cell populations

• This combined approach allows correlation between transcriptome profiles and protein localization

Patch-clamp electrophysiology integration:

• Record from individual neurons followed by immunostaining for SYNJ1

• This approach correlates SYNJ1 expression levels with electrophysiological properties

• Fixed biocytin-filled neurons can be processed for SYNJ1 immunofluorescence

Spatial transcriptomics combination:

• Technologies like Visium or MERFISH can map SYNJ1 mRNA distribution

• Follow with FITC-conjugated SYNJ1 antibody on adjacent sections

• This reveals relationships between transcription and translation/protein stability

Methodological workflow:

• Perform single-cell analysis to identify cells/regions of interest

• Apply SYNJ1 Antibody, FITC conjugated using optimized protocols

• Use image registration algorithms to align datasets

• Employ computational approaches to correlate different data modalities

This integrated approach is particularly valuable for understanding cell-type specific roles of SYNJ1 in complex neural tissues and disease states.

What techniques can be combined with SYNJ1 immunofluorescence to study phosphoinositide dynamics in neuronal systems?

For comprehensive analysis of SYNJ1-mediated phosphoinositide dynamics:

Live imaging approaches:

• Genetically-encoded PIP2 sensors (e.g., PH-PLCδ-GFP) can be combined with post-fixation SYNJ1 immunostaining

• This temporal-spatial correlation reveals how SYNJ1 localization relates to PIP2 dynamics

Super-resolution microscopy:

• STORM or STED microscopy of SYNJ1-FITC provides nanoscale localization

• Dual-color super-resolution imaging with clathrin or other endocytic proteins reveals precise spatial relationships

Biochemical phosphoinositide quantification:

• Combine SYNJ1 immunofluorescence with mass spectrometry analysis of phosphoinositides

• This correlates SYNJ1 distribution with actual lipid levels

FRET-based assays:

• Develop FRET sensors to monitor SYNJ1-substrate interactions

• This approach provides real-time visualization of enzymatic activity

Technical implementation:

• For dual PIP2/SYNJ1 immunofluorescence:

  • Use anti-PIP2 antibody (Echelon Z-P034) with SYNJ1 Antibody, FITC conjugated

  • Apply appropriate secondary antibodies (e.g., Alexa-647 for PIP2 detection)

  • Image using confocal microscopy with sequential scanning

• For electrophysiology/imaging combinations:

  • Perform patch-clamp recording with PIP2 modulation

  • Fix and stain for SYNJ1 localization

  • Correlate SYNJ1 distribution with electrophysiological responses