SYNJ2BP Antibody

What is SYNJ2BP and what are its primary cellular functions?

SYNJ2BP (Synaptojanin 2 Binding Protein) is a 16 kDa outer mitochondrial membrane protein with a cytosolic PDZ domain that functions as a cellular signaling hub . It plays a critical role in regulating mitochondria-ER membrane contact sites (MERC) . SYNJ2BP is essential for the formation of mitochondrion-associated endoplasmic reticulum membrane (MAM), specialized structures where mitochondria and ER membranes are in close contact .

These contact sites serve multiple functions:

• Facilitating calcium and ATP transfer between organelles

• Supporting oxidative protein production in the ER

• Regulating mitochondrial distribution within cells

• Controlling lipid metabolism, particularly in hepatocytes

Recent research has demonstrated that SYNJ2BP can improve the infectivity of lentiviruses by enhancing the production of viral envelope proteins through increased MAM formation .

What applications are SYNJ2BP antibodies validated for?

SYNJ2BP antibodies have been validated for multiple experimental applications:

For optimal results, each antibody should be titrated in your specific experimental system as performance can be sample-dependent .

What specifications should researchers consider when selecting a SYNJ2BP antibody?

When selecting a SYNJ2BP antibody, researchers should consider:

• Reactive species: Most commercial antibodies show reactivity with human, mouse, and rat samples

• Clonality: Both polyclonal and monoclonal options are available:

  • Polyclonal: Better for detecting low-abundance proteins and native epitopes

  • Monoclonal: Higher specificity and batch consistency

• Host species: Typically rabbit or mouse

• Application validation: Ensure the antibody is validated for your specific application

• Immunogen information: Different antibodies target different regions of SYNJ2BP:

  • Full-length (AA 1-145)

  • N-terminal region (AA 1-117)

  • Specific epitopes (AA 22-66)

• Storage conditions: Most SYNJ2BP antibodies require storage at -20°C and contain preservatives like sodium azide and glycerol

How should researchers optimize Western blot protocols for SYNJ2BP detection?

For optimal Western blot detection of SYNJ2BP:

• Sample preparation:

  • For mitochondrial proteins, consider mitochondrial enrichment protocols

  • Use RIPA buffer with protease inhibitors for whole cell lysates

  • Load 20-50 μg of total protein per lane

• Electrophoresis conditions:

  • Use 12-15% gels due to SYNJ2BP's small size (16 kDa)

  • Include positive control samples (e.g., mouse kidney or lung tissue)

• Transfer parameters:

  • Use PVDF membrane (0.2 μm pore size) for small proteins

  • Transfer at lower voltage (e.g., 30V overnight) for more efficient transfer

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Use primary antibody at 1:500-1:2000 dilution

  • Incubate at 4°C overnight for optimal signal

• Detection considerations:

  • Enhanced chemiluminescence (ECL) or fluorescent secondary antibodies

  • Expected band size: 16 kDa

  • Be aware of potential post-translational modifications

• Controls:

  • Include SYNJ2BP knockdown/knockout samples as negative controls

  • Consider using SYNJ2BP overexpression samples as positive controls

What methodologies are recommended for visualizing SYNJ2BP-mediated mitochondrial-ER contacts?

To effectively visualize and quantify SYNJ2BP-mediated mitochondrial-ER contacts:

• Transmission Electron Microscopy (TEM):

  • Gold standard for direct visualization of membrane contacts

  • Quantify contacts with <30 nm spacing between mitochondrial and ER membranes

  • Perform blinded analysis to prevent bias

  • Calculate the percentage of mitochondrial surface in contact with ER

• Proximity Ligation Assay (PLA):

  • Use antibodies against mitochondrial markers (e.g., Tom20) and ER markers (e.g., KDEL)

  • Detects protein-protein interactions in situ at distances <40 nm

  • Quantify PLA signal dots per cell

• Fluorescence microscopy approaches:

  • Dual-color immunofluorescence with mitochondrial and ER markers

  • Live-cell imaging with organelle-specific dyes or fluorescent proteins

  • Super-resolution microscopy for nanoscale resolution

• Quantitative analysis methods:

  • Colocalization analysis (Pearson's or Mander's coefficients)

  • Mahalanobis distance calculation for unbiased mitochondrial distribution analysis

  • Automated MATLAB scripts for quantification of organelle distribution

• Experimental controls:

  • Compare normal conditions, SYNJ2BP knockout, and SYNJ2BP overexpression

  • Include known MAM modulators as positive controls

In SYNJ2BP overexpression studies, researchers observed significant increases in mitochondria-ER contacts and perinuclear clustering of mitochondria in the soma, with diminished mitochondrial distribution in neurites .

What are the key considerations for SYNJ2BP knockdown and overexpression experiments?

When designing SYNJ2BP manipulation experiments:

For knockdown/knockout approaches:

• Method selection based on experimental needs:

  • siRNA: Achieves transient knockdown; effective in studies with eMDMs

  • shRNA (e.g., AAV8-sh-Synj2bp): Provides more stable knockdown; achieved 75% reduction in liver studies

  • CRISPR-Cas9: Generates complete knockout; successfully used in HEK293T cells

• Validation strategies:

  • Confirm knockdown at both mRNA (RT-PCR) and protein (Western blot) levels

  • Quantitative proteomics provides additional validation

  • Immunofluorescence confirms reduced expression at the cellular level

• Functional validation:

  • Assess changes in mitochondrial-ER contacts via TEM or PLA

  • Measure alterations in mitochondrial distribution

  • Evaluate impact on specific processes (viral replication, lipid metabolism, etc.)

For overexpression experiments:

• Expression system considerations:

  • Viral vectors for high-efficiency transduction

  • Inducible systems for controlled expression

  • Tagged constructs (with caution not to disrupt function)

• Expression level monitoring:

  • Establish dose-dependency (as observed with HIV Env production)

  • Compare physiological vs. high overexpression effects

• Localization confirmation:

  • Verify mitochondrial localization of overexpressed SYNJ2BP

  • Co-staining with organelle markers

• Essential controls:

  • Empty vector controls

  • Rescue experiments in knockout cells (successfully demonstrated in SYNJ2BP KO cells)

  • Wild-type vs. mutant domain constructs

Research has shown that SYNJ2BP reconstitution in knockout cells restores MAM formation and envelope protein production in lentiviral studies , confirming the specificity of observed effects.

How does SYNJ2BP contribute to lentiviral envelope protein production?

SYNJ2BP enhances lentiviral envelope (Env) protein production through several mechanisms:

• MAM formation and function:

  • SYNJ2BP increases mitochondrion-associated ER membrane formation

  • Enhanced MAMs provide more ATP and calcium ions to the ER, which are essential for Env protein folding and processing

  • MAMs reduce ER stress induced by HIV or EIAV Envs, increasing production levels

• Specificity of effect:

  • SYNJ2BP overexpression increases both EIAV and HIV Env protein levels

  • Effect is specific to Env proteins and does not affect structural proteins like Gag (p24/p26) or regulatory proteins like Rev

  • Effect is dose-dependent and reversible through rescue experiments

• Impact on viral infectivity:

  • Increased Env production correlates with enhanced viral infectivity

  • SYNJ2BP knockdown decreases viral replication efficiency

  • This effect has been demonstrated for both equine infectious anemia virus (EIAV) and HIV

• Mechanistic details:

  • Lentiviral Env proteins are rich in disulfide bonds and require proper folding in the ER

  • Misfolded Env proteins induce ER stress and are degraded by ER-associated protein degradation (ERAD)

  • SYNJ2BP-mediated MAM formation creates an optimal ER microenvironment for Env production

This research provides insights into previously unknown mechanisms regulating lentiviral Env production, which is critical for viral entry into host cells and could have implications for antiviral strategies .

What is the relationship between SYNJ2BP and motor neuron diseases?

SYNJ2BP has emerged as a significant factor in motor neuron diseases:

• Altered expression in motor neuron disorders:

  • Increased SYNJ2BP expression observed in iPSC-derived motor neurons from patients with spinal and bulbar muscular atrophy (SBMA)

  • Elevated SYNJ2BP protein levels in amyotrophic lateral sclerosis type 4 (ALS4) patient tissue

  • Confirmed by Western blot, RT-PCR, and immunohistochemistry

• Mechanisms of dysregulation:

  • In SBMA: Mutant androgen receptor (AR) with polyglutamine expansion binds near the SYNJ2BP gene promoter, enhancing expression

  • In ALS4: Protein levels increase without corresponding mRNA increase, suggesting altered protein turnover

  • SYNJ2BP expression increases in motor neurons under acute stress (e.g., H₂O₂ treatment), suggesting it may be part of stress response

• Functional consequences:

  • Increased mitochondria-ER membrane contacts in diseased motor neurons

  • Altered mitochondrial distribution with perinuclear clustering

  • Reduced mitochondrial presence in neurites/axons

  • Impaired mitochondrial oxidative function

• Therapeutic implications:

  • Reducing SYNJ2BP levels improves mitochondrial oxidative function in patient-derived motor neurons

  • Suggests SYNJ2BP as a potential therapeutic target in motor neuron diseases

• Research methodology:

  • Patient-derived iPSC motor neurons provide disease-relevant cellular models

  • Post-mortem spinal cord tissue confirms findings in human patients

  • Normalization to motor neuron marker (ChAT) accounts for neurodegeneration when quantifying SYNJ2BP levels

These findings identify SYNJ2BP as an important modulator of mitochondrial-ER contacts in motor neuron degeneration and suggest a common mechanism across different motor neuron diseases .

How does SYNJ2BP regulate hepatic lipid metabolism?

SYNJ2BP plays a crucial role in regulating hepatic lipid metabolism through its effects on mitochondria-ER contacts:

• Liver-specific SYNJ2BP function:

  • In mouse liver hepatocytes, SYNJ2BP regulates the extent of wrappER-mitochondria contacts

  • WrappER is a specialized wrapping-type ER rich in fatty acids that synthesizes lipoproteins (VLDL)

  • Nearly half of the surface area of every mitochondrion in hepatocytes is covered by wrappER

• Expression patterns:

  • SYNJ2BP expression in liver is comparable to other inter-organelle contact regulators like Mfn1/2 and VapA/B

  • Expression correlates with the extent of wrappER-mitochondria contacts

• Functional impact of SYNJ2BP manipulation:

  • Knockdown via AAV8-sh-Synj2bp reduces SYNJ2BP protein expression by 75% within three weeks

  • This leads to reduced wrappER-mitochondria contacts, as shown by quantitative electron microscopy

  • Results in altered fatty acid flux and hepatic lipid metabolism

• Effect on lipoprotein secretion:

  • SYNJ2BP knockdown causes a twofold increase in ApoB-100 expression (a VLDL marker)

  • Leads to approximately 25% increase in plasma triglycerides (but not cholesterol)

  • Suggests SYNJ2BP normally restricts lipoprotein secretion

This research demonstrates that SYNJ2BP is a key regulator of specialized mitochondria-ER contacts in the liver that control lipid metabolism and lipoprotein secretion .

How should researchers interpret contradictory SYNJ2BP data across different experimental systems?

When encountering contradictory SYNJ2BP data across different experimental systems, consider the following analytical framework:

By systematically addressing these factors, researchers can better reconcile apparently contradictory data and develop a more nuanced understanding of context-dependent SYNJ2BP functions.

How can researchers troubleshoot low SYNJ2BP signal in Western blots?

If experiencing weak SYNJ2BP signal in Western blots, systematically address these potential issues:

• Sample preparation optimization:

  • Enrich for mitochondrial fraction to concentrate SYNJ2BP

  • Use fresh samples with proper protease inhibitors

  • Avoid repeated freeze-thaw cycles of samples

  • Consider detergent selection (RIPA vs. NP-40 buffers)

• Protein transfer issues:

  • Use 0.2 μm PVDF membrane for better retention of small proteins

  • Modify transfer conditions for 16 kDa proteins (lower voltage, longer time)

  • Consider semi-dry transfer systems for small proteins

  • Verify transfer efficiency with reversible stains (Ponceau S)

• Antibody optimization:

  • Try higher antibody concentration (1:500 vs. 1:2000)

  • Extended primary antibody incubation (overnight at 4°C)

  • Test multiple SYNJ2BP antibodies targeting different epitopes

  • Ensure antibody reactivity matches your species (human/mouse/rat)

• Detection system enhancement:

  • Use high-sensitivity ECL substrates for chemiluminescence

  • Consider fluorescent secondary antibodies for improved quantification

  • Increase exposure time for weak signals

  • Try signal amplification systems if necessary

• Positive controls:

  • Include known positive samples (mouse kidney/lung tissue)

  • Consider running SYNJ2BP overexpression samples alongside

  • Use recombinant SYNJ2BP protein as reference

• Blocking optimization:

  • Try different blocking agents (milk vs. BSA)

  • Reduce blocking time if epitope accessibility is an issue

  • Consider specialized blocking buffers for phosphoprotein detection

• Expected results:

  • SYNJ2BP should appear as a distinct band at 16 kDa

  • Bands at other molecular weights may indicate non-specific binding or post-translational modifications

What are the critical factors for successful immunofluorescence detection of SYNJ2BP?

For optimal immunofluorescence detection of SYNJ2BP:

• Sample preparation:

  • Cell fixation: 4% paraformaldehyde preserves membrane structures

  • Permeabilization: Mild detergents (0.1-0.2% Triton X-100) for mitochondrial proteins

  • Antigen retrieval: Consider microwave-based methods for tissue sections

• Antibody selection and dilution:

  • Use antibodies validated for IF/ICC applications

  • Optimal dilution range: 1:200-1:800

  • Longer incubation (overnight at 4°C) may improve signal

• Co-staining strategy:

  • Include mitochondrial markers (MitoTracker, Tom20, COXIV)

  • Consider ER markers (PDI, calnexin, KDEL) for visualizing MAMs

  • Use high-quality secondary antibodies with minimal cross-reactivity

• Controls for validation:

  • SYNJ2BP knockdown/knockout cells as negative controls

  • Pre-adsorption of antibody with immunizing peptide

  • Secondary-only controls to assess background

• Image acquisition parameters:

  • Confocal microscopy for precise localization

  • Z-stack acquisition for complete cellular visualization

  • Consistent exposure settings across experimental conditions

• Quantification approaches:

  • Measure SYNJ2BP signal intensity at mitochondria

  • Assess colocalization with organelle markers

  • Analyze mitochondrial morphology and distribution

• Expected patterns:

  • Punctate mitochondrial localization

  • Enrichment at mitochondria-ER contact sites

  • Potential perinuclear clustering with overexpression

Successful SYNJ2BP immunofluorescence has been reported in multiple cell types including HepG2 cells and MCF-7 cells .

How can researchers validate the specificity of SYNJ2BP antibodies?

To ensure SYNJ2BP antibody specificity, implement these validation strategies:

• Genetic knockdown/knockout controls:

  • Test antibody in SYNJ2BP siRNA/shRNA-treated samples

  • Use CRISPR/Cas9 SYNJ2BP knockout cells as definitive negative controls

  • Compare signal reduction patterns across applications (WB, IF, IHC)

• Overexpression validation:

  • Test with tagged SYNJ2BP overexpression (but consider tag interference)

  • Verify signal increase correlates with expression level

  • Confirm proper subcellular localization

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide

  • Should abolish specific signal in all applications

  • Commercial blocking peptides are available for some antibodies

• Cross-validation with multiple antibodies:

  • Compare antibodies targeting different SYNJ2BP epitopes

  • Antibodies to N-terminal region (AA 1-117) vs. full-length (AA 1-145)

  • Consistent results across antibodies suggest specificity

• Mass spectrometry confirmation:

  • Immunoprecipitate with anti-SYNJ2BP and analyze by MS

  • Should identify SYNJ2BP and known interacting partners

  • Absence of unrelated proteins supports specificity

• Application-specific considerations:

  • Western blot: Single band at expected molecular weight (16 kDa)

  • IHC/IF: Signal pattern consistent with mitochondrial localization

  • IP: Enrichment of known interacting partners

• Species reactivity testing:

  • Confirm reactivity in claimed species (human, mouse, rat)

  • Test across multiple cell lines/tissues within each species

  • Be aware that antibody performance may vary between species

Multiple commercial SYNJ2BP antibodies have undergone validation in various applications across human, mouse, and rat samples .

How might SYNJ2BP be involved in neurodegenerative diseases beyond motor neuron disorders?

While SYNJ2BP's role in SBMA and ALS4 is documented , its potential involvement in other neurodegenerative diseases warrants investigation:

• Potential mechanisms relevant to broader neurodegeneration:

  • Mitochondrial dysfunction: A common feature across neurodegenerative diseases

  • Disrupted mitochondrial trafficking: Critical for neuronal health

  • Calcium dysregulation: SYNJ2BP-mediated MERC influences calcium homeostasis

  • ER stress responses: SYNJ2BP modulates ER stress, which is implicated in neurodegeneration

• Hypothesized roles in specific disorders:

  • Alzheimer's disease: Mitochondrial dysfunction and calcium dysregulation are key features

  • Parkinson's disease: PINK1-related mitophagy involves SYNJ2BP-regulated mRNA transport

  • Huntington's disease: Shares polyglutamine expansion mechanism with SBMA

  • Frontotemporal dementia: Involves disrupted mitochondrial dynamics

• Research approaches to explore these connections:

  • Analyze SYNJ2BP expression in patient-derived models and post-mortem tissues

  • Investigate genetic associations between SYNJ2BP variants and disease risk

  • Examine the effect of disease-associated proteins on SYNJ2BP expression and function

  • Develop conditional SYNJ2BP knockout models in specific neuronal populations

• Therapeutic implications:

  • SYNJ2BP modulation might represent a common therapeutic strategy across diseases

  • Target SYNJ2BP-mediated MAM formation to restore cellular homeostasis

  • Consider disease stage-specific interventions (early vs. late disease)

Future research should examine SYNJ2BP expression patterns across neurodegenerative diseases and investigate whether SYNJ2BP-targeted therapies might have broad neuroprotective effects.

What is the potential significance of SYNJ2BP in cancer research?

SYNJ2BP's functions suggest several potential roles in cancer biology that warrant investigation:

• Mitochondrial dynamics and cancer metabolism:

  • SYNJ2BP regulates mitochondrial distribution and function

  • Cancer cells often exhibit altered mitochondrial dynamics and metabolism

  • SYNJ2BP might influence metabolic reprogramming in tumors

• ER stress and unfolded protein response:

  • SYNJ2BP modulates ER stress through MAM regulation

  • Cancer cells frequently experience ER stress but adapt to avoid apoptosis

  • SYNJ2BP might affect cancer cell survival under stress conditions

• Calcium signaling in tumor progression:

  • MERC sites regulated by SYNJ2BP control calcium transfer

  • Calcium signaling affects proliferation, migration, and apoptosis in cancer

  • SYNJ2BP alterations could influence tumor growth and metastasis

• Lipid metabolism in cancer:

  • SYNJ2BP regulates hepatic lipid metabolism

  • Many cancers show altered lipid metabolism for membrane synthesis and signaling

  • SYNJ2BP might affect lipid-dependent processes in tumor cells

• Research approaches:

  • Analyze SYNJ2BP expression across cancer types and correlate with prognosis

  • Investigate functional consequences of SYNJ2BP knockdown/overexpression in cancer cell lines

  • Examine SYNJ2BP's role in tumor microenvironment and stress adaptation

  • Study how SYNJ2BP affects response to cancer therapies

Limited evidence from the search results shows that SYNJ2BP antibodies have been used to study human stomach cancer tissue , suggesting a potential role in gastric cancer that needs further exploration.

How might SYNJ2BP-targeted approaches be developed for therapeutic applications?

Based on SYNJ2BP's involvement in disease mechanisms, several therapeutic approaches could be developed:

• For motor neuron diseases:

  • Rationale: Reducing SYNJ2BP improves mitochondrial oxidative function in SBMA and ALS4 patient-derived motor neurons

  • Approaches:

    • Antisense oligonucleotides targeting SYNJ2BP mRNA

    • Small molecule inhibitors of SYNJ2BP-mediated MAM formation

    • CRISPR-based gene editing to normalize SYNJ2BP levels

• For metabolic disorders:

  • Rationale: SYNJ2BP knockdown alters hepatic lipid metabolism and increases lipoprotein secretion

  • Approaches:

    • Liver-directed SYNJ2BP modulators to regulate triglyceride levels

    • Targeted nanoparticles for hepatocyte-specific delivery

    • Small molecules that modulate SYNJ2BP activity rather than expression

• For viral infections:

  • Rationale: SYNJ2BP enhances lentiviral Env protein production and viral infectivity

  • Approaches:

    • SYNJ2BP inhibitors as novel antivirals

    • Targeting MAM formation to reduce viral envelope production

    • Combination approaches with existing antiviral therapies

• Technical considerations for therapeutic development:

  • Delivery challenges for targeting specific tissues

  • Potential off-target effects due to SYNJ2BP's role in multiple cellular processes

  • Need for tissue-specific approaches (e.g., CNS vs. liver)

  • Dosing strategies to achieve optimal SYNJ2BP modulation without complete inhibition

• Monitoring therapeutic efficacy:

  • SYNJ2BP antibodies for pharmacodynamic biomarker assessment

  • Measuring mitochondrial-ER contacts as functional readouts

  • Assessing downstream functional outcomes (mitochondrial function, lipid profiles)

This emerging field requires further research to validate SYNJ2BP as a therapeutic target and develop targeted approaches with acceptable safety profiles.