SYNJ1 Antibody

What are the key structural and functional characteristics of SYNJ1 protein?

Synaptojanin 1 (SYNJ1) is a polyphosphoinositide phosphatase expressed as two major alternatively spliced isoforms: 145 kDa (SJ145) and 170 kDa (SJ170). The protein plays critical roles in membrane trafficking and synaptic vesicle recycling. SYNJ1 contains a 5'-phosphatase domain that prominently regulates plasma membrane PI(4,5)P₂ levels, as demonstrated by the increased membrane PI(4,5)P₂ observed in SYNJ1-deficient cells . The protein is primarily known for its neuronal functions, but research has revealed its expression in other cell types like astrocytes, where it appears to repress basal autophagosome formation . SYNJ1 mutations have been linked to Parkinsonism with seizures, highlighting its significance in neurological function .

What isoforms of SYNJ1 are recognized by commonly available antibodies?

Most commercially available SYNJ1 antibodies are designed to recognize multiple isoforms. For instance, the polyclonal antibody ABIN1742317 recognizes isoforms 1, 2, and 3 of synaptojanin 1, with specific binding to amino acids 1140-1155 in isoform 2 . The Proteintech antibody 24677-1-AP detects SYNJ1 with an observed molecular weight of approximately 140 kDa . When selecting an antibody, researchers should verify which isoforms are recognized based on the immunogen sequence and validation data. This is particularly important for experiments aimed at distinguishing between the neuronal isoform (145 kDa) and the 170 kDa isoform, as their differential expression may have distinct biological implications in various cell types and experimental conditions .

What are the recommended storage conditions for maintaining SYNJ1 antibody activity?

For optimal preservation of SYNJ1 antibody activity, store the antibodies at -20°C, where they remain stable for approximately one year after shipment . Lyophilized antibodies should never be stored in the freezer as this may destroy their activity; they can be stored at 4°C for several weeks or even months without significant loss of quality . After reconstitution, affinity-purified antibodies like ABIN1742317 should not be stored at 4°C for prolonged periods (several weeks) as they contain fewer protease inhibitors than antisera, making them less robust . For reconstitution of lyophilized formats, add the recommended volume (e.g., 50 μL H₂O for ABIN1742317) to achieve the desired concentration (1 mg/mL) in PBS, then aliquot and store at -20°C until use . Some formulations contain 0.02% sodium azide and 50% glycerol at pH 7.3 to enhance stability .

What are the optimal dilution ratios for different applications of SYNJ1 antibodies?

The optimal dilution ratios for SYNJ1 antibodies vary significantly depending on the specific application and the antibody preparation. For Western blotting (WB), the recommended dilution range for ABIN1742317 is 1:200 to 1:2000 for alkaline phosphatase (AP) staining , while Proteintech's 24677-1-AP can be used at dilutions between 1:1000 and 1:8000 . For immunohistochemistry (IHC), the effective dilution for 24677-1-AP ranges from 1:50 to 1:500 , and ABIN1742317 can be used at 1:1000 to 1:5000 . For immunofluorescence (IF) or immunocytochemistry (ICC), 24677-1-AP works well at dilutions between 1:20 and 1:200 . It is strongly recommended to titrate each antibody in your specific testing system, as optimal dilutions can be highly sample-dependent and may require adjustment based on expression levels, tissue type, and detection methods .

How should samples be prepared for optimal SYNJ1 detection by Western blotting?

For optimal detection of SYNJ1 by Western blotting, an important technical note is that running SDS-PAGE with non-boiled samples gives stronger signals compared to standard protocols . This suggests that SYNJ1 protein structure may be sensitive to heat denaturation, potentially affecting epitope accessibility. When using antibodies like ABIN1742317, prepare lysates in standard SDS-PAGE loading buffer but omit the boiling step . For tissue samples, mouse and rat brain tissues have been validated for successful detection with both ABIN1742317 and 24677-1-AP antibodies . Cell lines such as NIH/3T3 have also been successfully used for SYNJ1 detection . Prepare protein extracts using standard cell lysis buffers containing protease inhibitors to prevent degradation. For quantitative analysis, ensure equal loading (15-30 μg total protein per lane) and include appropriate housekeeping protein controls. The observed molecular weight for SYNJ1 detection is approximately 140 kDa , although the calculated molecular weight is 178 kDa (1612 amino acids) .

What tissue fixation and antigen retrieval methods work best for SYNJ1 immunohistochemistry?

For immunohistochemical detection of SYNJ1, the choice of fixation and antigen retrieval methods significantly impacts staining quality. The Proteintech antibody 24677-1-AP has been successfully used on human and rat brain and skeletal muscle tissues . For optimal results with this antibody, antigen retrieval with TE buffer at pH 9.0 is recommended . Alternatively, citrate buffer at pH 6.0 can be used for antigen retrieval if the TE buffer method proves suboptimal for specific tissue preparations . When working with ABIN1742317 for IHC applications, use at dilutions between 1:1000 and 1:5000 . The fixation method should be selected based on the specific tissue being examined - for brain tissue samples, 4% paraformaldehyde fixation followed by paraffin embedding is commonly used. For frozen sections, brief fixation in ice-cold acetone or methanol may be preferred to preserve antigenicity. Always include positive control tissues (rat or mouse brain) for which the antibodies have been validated to confirm staining specificity .

How can researchers verify SYNJ1 antibody specificity in their experimental systems?

Verifying SYNJ1 antibody specificity requires multiple validation approaches. First, perform Western blotting using positive controls known to express SYNJ1, such as mouse or rat brain tissue, alongside negative controls like cell types with minimal SYNJ1 expression . The antibody should detect bands at the expected molecular weights (approximately 140-145 kDa for the neuronal isoform) . For definitive validation, compare staining patterns between wild-type tissues/cells and those with SYNJ1 knockout or knockdown - the signal should be absent or significantly reduced in the latter . Additionally, pre-absorption tests can be performed by incubating the antibody with its immunogenic peptide (e.g., CGVGAPPSPGVTRREME for ABIN1742317) prior to application . If the antibody is specific, this should eliminate or substantially reduce staining. For immunohistochemistry or immunofluorescence applications, the staining pattern should be consistent with known SYNJ1 localization and should vary appropriately across different tissues (strong in neuronal tissues, weaker in astrocytes) . When using multiple antibodies targeting different epitopes of SYNJ1, similar staining patterns provide additional confidence in specificity.

What are the common technical challenges in SYNJ1 Western blotting and how can they be resolved?

When performing Western blotting for SYNJ1, researchers may encounter several technical challenges. One common issue is weak signal strength, which can be addressed by running SDS-PAGE with non-boiled samples, as this has been shown to give stronger signals for SYNJ1 detection . If bands appear at unexpected molecular weights, verify sample preparation methods and consider that SYNJ1 has multiple isoforms (145 kDa and 170 kDa) that may be detected differently . High background can be reduced by optimizing blocking conditions (try 5% non-fat milk or BSA) and increasing washing steps. If multiple bands appear, they may represent degradation products or cross-reactivity; validate using SYNJ1-deficient samples as negative controls . For faint bands with the neuronal 145 kDa isoform in non-neuronal tissues like astrocytes, longer exposure times may be necessary, as SYNJ1 is expressed at low levels in these cell types . When comparing SYNJ1 levels between wild-type and knockout/knockdown samples, ensure equal loading by using reliable housekeeping proteins and quantify bands using appropriate software to normalize SYNJ1 signal to loading controls.

How can researchers differentiate between specific and non-specific staining patterns in SYNJ1 immunofluorescence?

Differentiating between specific and non-specific staining in SYNJ1 immunofluorescence requires careful analysis and appropriate controls. Specific SYNJ1 staining should correspond to its known subcellular localization - primarily cytoplasmic with possible membrane association . In neurons, enrichment at synaptic terminals would be expected, while in astrocytes, a more diffuse cytoplasmic pattern might be observed . To identify non-specific binding, include negative controls such as primary antibody omission and isotype controls (rabbit IgG for polyclonal antibodies like 24677-1-AP) . The staining should be significantly reduced in SYNJ1-deficient or knockdown cells compared to wild-type cells . When evaluating staining patterns in NIH/3T3 cells (a validated cell line for 24677-1-AP), compare your results with published patterns . For co-localization studies, use confirmed markers of subcellular compartments to verify appropriate SYNJ1 localization. Specific staining should be concentration-dependent - serial dilutions of the antibody should result in proportional reductions in signal intensity while maintaining the same pattern. Non-specific staining often appears as uniform background or random spots that don't change with antibody dilution or in knockout controls.

How can SYNJ1 antibodies be utilized to investigate autophagy dysregulation in neurodegenerative disorders?

SYNJ1 antibodies serve as valuable tools for investigating autophagy dysregulation in neurodegenerative conditions, given the emerging role of SYNJ1 in autophagosome formation. Research has established that SYNJ1 represses basal autophagosome formation, with SYNJ1-deficient astrocytes exhibiting hyperactive autophagosome production, characterized by increased size and number of GFP-LC3 structures . To leverage SYNJ1 antibodies in autophagy studies, researchers should implement dual-marker approaches: combine SYNJ1 immunostaining with autophagy markers such as LC3-II and p62, which are elevated in aged SYNJ1+/- mouse brain lysates . Co-immunoprecipitation (co-IP) using SYNJ1 antibodies can identify protein interaction partners that connect SYNJ1 to the autophagy machinery. The 24677-1-AP antibody has been validated for co-IP applications . For examining dynamic changes, compare autophagosome formation in the presence or absence of bafilomycin A1 (20 nM for 1 hour), which inhibits autolysosomal degradation and amplifies differences between wild-type and SYNJ1-deficient cells . SYNJ1 antibodies can also be employed to investigate the relationship between SYNJ1's phosphatase activity and autophagy by correlating PI(4,5)P₂ levels with autophagosome formation, as SYNJ1 deletion results in increased plasma membrane PI(4,5)P₂ .

What experimental approaches can detect SYNJ1 expression in non-neuronal cells like astrocytes?

Detecting SYNJ1 expression in non-neuronal cells like astrocytes requires sensitive techniques due to its relatively low expression levels compared to neurons . Western blotting using highly sensitive detection methods represents a fundamental approach - the Novus antibody specific for the Synj1-145 kDa neuronal isoform has successfully identified low-level expression in cultured cortical astrocytes but not in microglia or HEK293T cells . For immunofluorescence detection, use higher antibody concentrations within the recommended range (closer to 1:20 than 1:200 for 24677-1-AP) and employ signal amplification techniques such as tyramide signal amplification (TSA). Co-staining with astrocyte-specific markers (GFAP, S100β, or ALDH1L1) helps confirm cell identity. Quantitative PCR offers another sensitive method to detect SYNJ1 mRNA expression in purified astrocyte populations. Single-cell RNA sequencing can reveal cell-type-specific expression patterns of SYNJ1 in heterogeneous brain tissue. For functional studies, compare SYNJ1 knockout/knockdown effects in astrocytes to those in neurons, focusing on endpoints like PI(4,5)P₂ levels, which are elevated in SYNJ1-deficient astrocytes similar to neurons , and GluT1 levels, which are consistently reduced at the membrane of both SYNJ1+/- and SYNJ1-/- astrocytes .

How can SYNJ1 antibodies be applied to study the relationship between SYNJ1 and glucose transporter regulation?

SYNJ1 antibodies can be instrumental in exploring the newly discovered relationship between SYNJ1 and glucose transporter regulation. Research has shown a consistent and significant reduction of the astrocyte-specific glucose transporter GluT1 at the membrane of both SYNJ1+/- and SYNJ1-/- astrocytes across multiple independent culture batches . To investigate this relationship, researchers should employ cell fractionation techniques followed by Western blotting with SYNJ1 antibodies (such as 24677-1-AP at 1:1000-1:8000 dilution) and GluT1 antibodies to quantify their relative levels in membrane versus cytosolic fractions. Co-immunoprecipitation experiments using SYNJ1 antibodies can identify potential direct interactions between SYNJ1 and components of glucose transporter trafficking machinery. For visualizing spatial relationships, perform dual immunofluorescence labeling of SYNJ1 and GluT1, using 24677-1-AP antibody at appropriate dilutions (1:20-1:200) . To establish causality, conduct rescue experiments in SYNJ1-deficient cells by expressing wild-type SYNJ1 and monitoring GluT1 membrane localization. Since AMPK activity is enhanced at basal levels in SYNJ1-deficient astrocytes , researchers should investigate whether SYNJ1 regulates GluT1 through AMPK-dependent pathways by combining SYNJ1 immunodetection with phospho-AMPK antibodies in parallel experiments. This approach can help determine if SYNJ1's role in glucose transport is related to its effects on autophagy, as both processes appear dysregulated in SYNJ1-deficient cells .

What methodologies can be used to investigate the relationship between SYNJ1 and PI(4,5)P₂ regulation in different cellular compartments?

Investigating SYNJ1's relationship with PI(4,5)P₂ across different cellular compartments requires sophisticated methodological approaches. Immunofluorescence co-localization studies using 24677-1-AP SYNJ1 antibody (1:20-1:200) alongside PI(4,5)P₂-specific antibodies or fluorescent PI(4,5)P₂ biosensors (like PH-PLCδ-GFP) can map their spatial relationship. For subcellular fractionation, isolate different membrane compartments (plasma membrane, endosomes, autophagosomes) and analyze SYNJ1 and PI(4,5)P₂ levels within each fraction using Western blotting with antibodies like ABIN1742317 (1:200-1:2000) . Live-cell imaging with SYNJ1-fluorescent protein fusions combined with PI(4,5)P₂ biosensors allows real-time monitoring of their dynamic interactions. To assess functional relationships, compare PI(4,5)P₂ levels in wild-type versus SYNJ1-deficient cells across multiple compartments - deletion of SYNJ1 results in increased plasma membrane PI(4,5)P₂, suggesting the prominence of its 5'-phosphatase domain in membrane signaling . Structure-function studies can be conducted by expressing mutant SYNJ1 proteins with inactivated phosphatase domains and immunoprecipitating with SYNJ1 antibodies to determine which domains are required for proper PI(4,5)P₂ regulation. For in vitro phosphatase assays, immunoprecipitate SYNJ1 using antibodies validated for IP applications and measure its activity against PI(4,5)P₂ substrates.

How can comparative immunostaining be optimized when studying SYNJ1 in both wild-type and gene-modified models?

Optimizing comparative immunostaining when studying SYNJ1 in wild-type and gene-modified models requires rigorous experimental design and careful validation. First, establish a standardized tissue processing workflow - all samples should undergo identical fixation, antigen retrieval, and staining protocols. For SYNJ1 detection in brain tissues, use TE buffer at pH 9.0 for antigen retrieval as recommended for 24677-1-AP antibody . Process wild-type and gene-modified samples in parallel on the same slides whenever possible to minimize technical variations. Titrate antibody concentrations specifically for comparative studies - determine the linear detection range where signal intensity correlates with protein abundance without reaching saturation. For SYNJ1 antibody 24677-1-AP, test multiple dilutions within the recommended range (1:50-1:500 for IHC) . Include internal control tissues or regions known to express constant SYNJ1 levels regardless of genotype for signal normalization. When staining cultured cells, co-stain for cell-type markers to ensure comparison of equivalent cell populations. For quantitative analysis, use automated imaging platforms and analysis software with standardized acquisition settings and blinded analysis procedures. Validate immunostaining results with complementary techniques like Western blotting, where non-boiled samples provide stronger SYNJ1 signals . When comparing results across batches, include standard reference samples in each batch to permit normalization.