SYNJ2 Antibody

What is SYNJ2 and why is it important in research?

SYNJ2 (Synaptojanin-2) is an inositol polyphosphate phosphatase that plays significant roles in recycling neurotransmitter vesicles and is implicated in spermatogenesis . Also known as KIAA0348, it belongs to the synaptojanin family and has essential functions in distinct membrane trafficking and signal transduction pathways . SYNJ2 may mediate the inhibitory effect of Rac1 on endocytosis, making it an important target for neurological and cell biology research . The protein has three isoforms produced by alternative splicing, with a calculated molecular weight of 166 kDa, though it is frequently observed at 140-160 kDa in experimental conditions . Recent research has also suggested potential associations between SYNJ2 and colorectal cancer risk, based on studies of the rs9365723 polymorphism in Chinese populations .

What are the key characteristics of commercially available SYNJ2 antibodies?

Most commercial SYNJ2 antibodies are polyclonal antibodies raised in rabbits, with reactivity against human, mouse, and rat samples . These antibodies typically target specific regions of the SYNJ2 protein, such as the central region (amino acids 679-707) or other epitopes derived from human SYNJ2 . They are generally supplied in liquid form with storage buffers containing PBS, sodium azide, and glycerol for stability . The antibodies undergo purification processes such as antigen affinity chromatography or protein A column purification followed by peptide affinity purification to ensure specificity . They are delivered unconjugated but can be used in various detection methods including Western blot, immunohistochemistry, immunofluorescence, and ELISA applications .

What is the recommended storage and handling procedure for SYNJ2 antibodies?

SYNJ2 antibodies should be stored at -20°C for optimal stability and longevity . Most manufacturers indicate that the antibodies remain stable for at least one year after receipt when stored properly . For antibodies supplied in larger volumes, aliquoting is recommended to avoid repeated freeze/thaw cycles that can compromise antibody integrity and performance . Some formulations (particularly those containing 50% glycerol) may not require aliquoting for -20°C storage . If small volumes become entrapped in the vial's seal during shipping or storage, brief centrifugation in a tabletop centrifuge can dislodge the liquid . Prior to use, allow the antibody to equilibrate to room temperature, and gently mix before pipetting to ensure homogeneity of the solution .

How should SYNJ2 antibodies be optimized for Western blot applications?

For Western blot applications, SYNJ2 antibodies typically require optimization within specific dilution ranges, generally between 1:500-1:3000 depending on the manufacturer and specific antibody clone . The optimization process should begin with a middle-range dilution and adjust based on signal strength and background levels. When detecting SYNJ2, researchers should be prepared to observe bands around 140-160 kDa, which may vary slightly from the calculated molecular weight of 166 kDa . Cell lines that have shown positive Western blot results with SYNJ2 antibodies include HEK-293 cells and Jurkat cells, making these good positive controls for initial optimization experiments . For optimal results, use freshly prepared protein samples and include appropriate blocking agents to minimize non-specific binding. Validation of antibody specificity can be conducted using knock-down or knock-out controls when available to confirm the identity of the detected bands .

What are the critical parameters for successful immunohistochemistry (IHC) with SYNJ2 antibodies?

For immunohistochemistry applications, SYNJ2 antibodies are typically used at dilutions ranging from 1:20 to 1:200, requiring more concentrated antibody solutions than Western blot applications . Antigen retrieval is critical for successful SYNJ2 detection in fixed tissues, with recommended protocols including TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 . Positive IHC detection has been reported in human brain tissue and mouse small intestine tissue, which can serve as appropriate positive controls . For optimal staining, tissue sections should be appropriately fixed (typically with 4% paraformaldehyde) and permeabilized before antibody incubation. Background reduction strategies including proper blocking with serum matching the secondary antibody species are essential. Incubation times and temperatures should be optimized based on tissue type and thickness, with overnight incubations at 4°C often yielding the best signal-to-noise ratio for this antibody .

What protocol considerations are important for immunofluorescence (IF) staining with SYNJ2 antibodies?

For immunofluorescence applications, SYNJ2 antibodies are typically used at dilutions between 1:200 and 1:800 . SH-SY5Y cells have been validated for positive IF/ICC detection of SYNJ2, making them an appropriate positive control for these applications . When performing IF with SYNJ2 antibodies, key protocol considerations include proper fixation (4% paraformaldehyde for 15-20 minutes at room temperature is typically effective), adequate permeabilization (0.1-0.3% Triton X-100 for 5-10 minutes), and thorough blocking (5% normal serum from the same species as the secondary antibody, for at least 1 hour). Antibody incubation times should be optimized, with primary antibody incubation often yielding best results when performed overnight at 4°C. Washing steps should be thorough (3-5 washes of 5-10 minutes each) to reduce background. When imaging, it's important to include appropriate controls including secondary-only controls to assess background fluorescence and, when possible, known positive and negative samples for comparison .

How can researchers resolve discrepancies between observed and predicted molecular weights of SYNJ2?

The calculated molecular weight of SYNJ2 is approximately 166 kDa, but observed molecular weights in experimental settings often range from 140-160 kDa . This discrepancy is not uncommon and may arise from several factors. First, post-translational modifications such as phosphorylation, glycosylation, or proteolytic processing can alter the apparent molecular weight of proteins on SDS-PAGE gels . Second, the presence of multiple isoforms (SYNJ2 has three known isoforms produced by alternative splicing) may result in detection of bands at different molecular weights . To resolve these discrepancies, researchers should:

• Use gradient gels (4-15% or 4-20%) to improve separation of high molecular weight proteins

• Include phosphatase or glycosidase treatments in parallel samples to determine if post-translational modifications contribute to the observed weight differences

• Use isoform-specific antibodies or primers when available to determine which specific isoform is being detected

• Compare results across multiple detection methods (e.g., mass spectrometry) to confirm the identity of the observed protein bands

What strategies can overcome cross-reactivity issues when working with SYNJ2 antibodies?

Cross-reactivity can be a significant challenge when working with SYNJ2 antibodies, particularly given structural similarities with other synaptojanin family members. To minimize cross-reactivity issues, researchers should implement several strategies:

• Increase antibody dilution: Begin by testing more dilute antibody concentrations to reduce non-specific binding while maintaining specific signal detection

• Optimize blocking conditions: Use 5% BSA or normal serum from the same species as the secondary antibody in TBS-T, and extend blocking time to 2 hours at room temperature

• Increase washing frequency and duration: Implement 5-6 washes of 10 minutes each with TBS-T containing 0.1-0.3% Tween-20

• Pre-adsorb the antibody: When possible, pre-incubate the diluted antibody with tissues or cells known to lack SYNJ2 expression to remove antibodies with affinity for other proteins

• Include competitive peptide blocking: Use the immunizing peptide to confirm specificity, where signal abolishment upon peptide competition indicates specific binding

• Validate with multiple antibodies: Use antibodies targeting different epitopes of SYNJ2 to confirm consistent detection patterns

How should researchers design experiments to study SYNJ2 interactions with binding partners?

SYNJ2 interacts with several proteins, including GRB2, and its isoforms have specific binding patterns (e.g., isoform 2A binds to SYNJ2BP/OMP25, and isoform 2B2 binds to SH3 domain-containing proteins) . When designing experiments to study these interactions, researchers should consider the following methodological approaches:

• Co-immunoprecipitation (Co-IP): Use SYNJ2 antibodies for pull-down experiments followed by immunoblotting for suspected binding partners. For optimal results:

  • Use mild lysis buffers containing 1% NP-40 or 0.5% Triton X-100 to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors in all buffers

  • Consider crosslinking strategies for transient interactions

• Proximity ligation assay (PLA): This technique can visualize protein interactions in situ with high sensitivity:

  • Use antibodies raised in different species for SYNJ2 and its potential binding partner

  • Optimize fixation conditions to preserve cellular structures

  • Include appropriate controls (single antibody controls and known interaction pairs)

• FRET or BRET analyses: For real-time interaction studies in living cells:

  • Create appropriate fusion constructs with fluorescent or luminescent tags

  • Validate that tags do not interfere with protein function

  • Control for expression levels to avoid artifacts from overexpression

How can SYNJ2 antibodies be applied in neuroscience research?

SYNJ2 functions in recycling neurotransmitter vesicles, making it highly relevant to neuroscience research . SYNJ2 antibodies can be utilized in several neuroscience applications:

• Synaptic vesicle trafficking studies: Use immunofluorescence with SYNJ2 antibodies to visualize its localization during various stages of the synaptic vesicle cycle in primary neuronal cultures. Combine with other synaptic markers (e.g., synaptophysin, synaptotagmin) to analyze colocalization patterns during stimulation and recovery phases.

• Brain region expression mapping: Apply immunohistochemistry with SYNJ2 antibodies to brain sections to map region-specific expression patterns. Human brain tissue has been validated for SYNJ2 antibody staining, allowing for comparative studies across species and in various neurological conditions .

• Neurological disorder investigations: Compare SYNJ2 expression and localization in tissue samples from patients with neurological disorders versus healthy controls. SYNJ2 has been associated with cognition disorders in published literature, suggesting potential relevance to cognitive dysfunction research .

• Neuronal activity studies: Combine SYNJ2 immunostaining with activity markers to investigate how neuronal activation alters SYNJ2 distribution and phosphorylation state, potentially revealing mechanisms of activity-dependent synaptic plasticity .

What role does SYNJ2 play in cancer research, and how can antibodies facilitate these studies?

Research has suggested potential associations between SYNJ2 and cancer, including colorectal cancer and prostatic neoplasms . SYNJ2 antibodies can be valuable tools in cancer research through several approaches:

• Expression analysis in tumor samples: Use immunohistochemistry with SYNJ2 antibodies to compare expression levels between tumor and adjacent normal tissues. This can help establish whether SYNJ2 is upregulated or downregulated in specific cancer types and correlate with clinical parameters.

• Signal transduction pathway studies: Since SYNJ2 functions in phosphoinositide signaling pathways, which are frequently dysregulated in cancer, antibodies can help track SYNJ2's role in these pathways:

  • Use phospho-specific antibodies (when available) to monitor SYNJ2 activation state

  • Combine with inhibitors of upstream regulators to map signaling hierarchies

• Migration and invasion assays: Given SYNJ2's role in membrane trafficking and potential connection to cytoskeletal dynamics, antibodies can be used to:

  • Track subcellular localization during cell migration

  • Evaluate colocalization with focal adhesion markers

  • Assess changes in response to migration-promoting stimuli

• Genetic association validation: For SNPs like rs9365723 that have been associated with cancer risk, antibodies can help determine if these genetic variations correlate with altered protein expression or localization in patient-derived samples .

How can SYNJ2 antibodies be utilized in studying phosphoinositide signaling pathways?

SYNJ2 functions as an inositol polyphosphate phosphatase involved in several phosphoinositide signaling pathways, including 3-phosphoinositide degradation, D-myo-inositol trisphosphate biosynthesis, and inositol phosphate metabolism . SYNJ2 antibodies can facilitate research in these pathways through:

• Pathway activation studies: Use SYNJ2 antibodies in Western blot analyses to monitor changes in SYNJ2 expression or phosphorylation following stimulation of cells with growth factors or inhibitors that modulate phosphoinositide signaling. This can reveal how SYNJ2 responds to and regulates these pathways.

• Subcellular localization analyses: Employ immunofluorescence microscopy with SYNJ2 antibodies to:

  • Track dynamic relocalization of SYNJ2 during signaling events

  • Co-stain with markers of specific membrane compartments (e.g., PIP2, PIP3 sensors)

  • Visualize changes in response to pathway inhibitors

• Substrate specificity investigations: Combine immunoprecipitation using SYNJ2 antibodies with in vitro phosphatase assays to:

  • Determine preferred phosphoinositide substrates

  • Measure activity changes in response to cellular stimuli

  • Identify regulatory post-translational modifications

• Multi-protein complex analyses: Use SYNJ2 antibodies in combination with antibodies against other pathway components to investigate how signaling complexes form and dissolve during pathway activation and termination .

What are the optimal conditions for different experimental applications of SYNJ2 antibodies?

Different experimental applications require specific optimization of SYNJ2 antibody conditions. The table below summarizes recommended dilutions and conditions for key applications:

This comparison highlights the need to adjust antibody concentration based on application, with IHC typically requiring more concentrated antibody solutions than Western blot or immunofluorescence . Each application benefits from specific positive controls that have been validated for SYNJ2 detection, ensuring reliable experimental outcomes.