SYNPO Antibody

What is Synaptopodin and why is it important in research?

Synaptopodin (SYNPO) is a proline-rich actin-associated protein highly expressed in telencephalic dendrites and renal podocytes. It represents a novel class of actin-binding proteins with two binding sites for actin . Its significance in research stems from its dual localization pattern and role in:

• Neuronal tissue: Found in a subset of exclusively telencephalic synapses, with differentiation-dependent expression during postnatal brain maturation

• Kidney tissue: Expressed in differentiated podocytes (glomerular visceral epithelial cells), where it colocalizes with alpha-actinin

Functionally, SYNPO plays a protective role in podocyte injury through its involvement in actin reorganization and focal adhesion dynamics, making it a critical protein for studying kidney diseases like focal segmental glomerulosclerosis (FSGS) .

What are the different isoforms of SYNPO and how can they be detected?

SYNPO exists in multiple isoforms with distinct molecular weights and tissue distributions:

Detection methods vary by isoform:

• The neural form (100 kDa) is detectable in brain tissue using antibodies like the polyclonal NT antibody

• The renal form (110 kDa) requires antibodies specific to Synpo-long, such as those targeting the Synpo-alt fragment (AA 671–903)

• Some antibodies can detect both isoforms, while others are isoform-specific, requiring careful selection based on experimental needs

What are the optimal storage conditions for SYNPO antibodies?

Proper storage of SYNPO antibodies is critical for maintaining reactivity and specificity. Based on manufacturer recommendations:

Most manufacturers recommend storing antibodies in PBS buffer containing 0.02-0.09% sodium azide and sometimes 50% glycerol (pH 7.3) to maintain stability . Centrifuge prior to opening is recommended for some formulations .

How should I optimize fixation and antigen retrieval for SYNPO immunohistochemistry?

Successful SYNPO immunohistochemistry requires appropriate fixation and antigen retrieval protocols:

Fixation methods:

• Methanol/acetone fixation: Immerse slides in precooled (-20°C) methanol for 5 minutes, followed by precooled (-20°C) acetone for 30-60 seconds, then air dry before antibody incubation

• Enhanced permeabilization: After methanol/acetone fixation, dip slides in 0.1-0.2% Triton X-100 in PBS or 0.1% saponin in PBS for 1-5 minutes at room temperature to improve accessibility of cytoskeletal antigens

Antigen retrieval recommendations:

• For paraffin-embedded tissues: Microwave treatment is recommended for most SYNPO antibodies

• Buffer options: TE buffer pH 9.0 is suggested as primary choice, with citrate buffer pH 6.0 as an alternative

Dilution ranges for different applications:

• IHC (frozen sections): 1:50-1:200

• IHC (paraffin sections): 1:20-1:3000 (varies by antibody and manufacturer)

• IF/ICC: 1:200-1:800

Why do I observe different molecular weights for SYNPO in Western blot analysis?

The variable molecular weights observed in Western blot analysis of SYNPO can be attributed to several factors:

• Tissue-specific isoforms:

  • Brain tissue typically shows a 100 kDa polypeptide (neural form)

  • Kidney tissue shows a 110 kDa polypeptide (renal form)

• Post-translational modifications:
  The difference between calculated and observed molecular weights is attributed to post-translational modifications. Calculated weights from sequence data are:

  • Human: 73.7 kDa (pI 9.38)

  • Mouse: 74 kDa (pI 9.27)

  Yet SDS-PAGE consistently shows higher molecular weights (100-110 kDa).

• Protein degradation:

  • A 44-45 kDa band often represents a degradation fragment of SYNPO

  • Proper sample preparation with protease inhibitors can minimize degradation

When troubleshooting Western blot variations, consider antibody specificity, tissue source, extraction methods, and running conditions as variables affecting observed molecular weights.

How do I select the appropriate SYNPO antibody for my specific research application?

Selecting the right SYNPO antibody requires consideration of several factors:

Epitope specificity:

• N-terminal antibodies (e.g., 03-GP94-N): Recognize both Synpo-S and Synpo-L isoforms

• C-terminal antibodies (e.g., 03-GP94-C): Recognize only Synpo-L and Synpo-T isoforms

• Internal region antibodies (e.g., 03-GP94-IN): Recognize both Synpo-S and Synpo-L

Host species considerations:

• Mouse monoclonal: High specificity, good for applications requiring consistent lot-to-lot reproducibility

• Rabbit polyclonal: Often provides stronger signals, good for detecting low-abundance proteins

• Guinea pig polyclonal: Useful for co-labeling experiments with mouse or rabbit primary antibodies

Application-specific selection matrix:

Cross-reference the antibody's tested reactivity with your experimental species to ensure compatibility .

What methodological approaches can confirm SYNPO-α-actinin interactions in experimental systems?

The interaction between SYNPO and α-actinin can be confirmed through multiple complementary approaches:

Co-immunoprecipitation (Co-IP) strategies:

• Endogenous protein Co-IP:

  • Precipitate endogenous proteins from podocyte extracts using anti-Synpo-alt antibody and anti-α-actinin-4 antibody

  • Western blot analysis confirms that anti-Synpo-alt precipitates Synpo-long and co-precipitates α-actinin-4

  • Conversely, anti-α-actinin-4 co-immunoprecipitates Synpo-long

• Tagged protein Co-IP in heterologous expression systems:

  • Co-express FLAG-α-actinin-4 with GFP-Synpo-long or GFP-Synpo-short in HEK293 cells

  • Both GFP-tagged SYNPO isoforms co-precipitate with FLAG-α-actinin-4

  • In complementary experiments, GFP-α-actinin-4 co-precipitates with FLAG-Synpo-alt and FLAG-Synpo-short

Yeast two-hybrid screening:

• Use Synpo-alt fragment (AA 671–903) fused to GAL4 DNA binding domain as bait

• Screen human kidney cDNA library

• Identify interacting proteins (e.g., α-actinin-4 and α-actinin-1 were identified in this manner)

Controls for interaction specificity:

• Include irrelevant control antibodies for immunoprecipitations

• Use GFP alone as a negative control for co-precipitation experiments

• Confirm absence of interaction with unrelated proteins

These methodological approaches provide complementary evidence for SYNPO-α-actinin interaction and help elucidate the functional significance of this interaction in podocyte biology and disease states like FSGS.

How can I validate SYNPO knockout models for functional studies?

Validating SYNPO knockout models requires a multi-faceted approach:

Generation strategies:

• CRISPR/Cas9-mediated deletion: Target guide RNAs to coding exons (e.g., exons 2 and 3 in mice)

• Design primers to amplify the deleted segment, confirming successful deletion

Validation at genomic level:

• PCR screening with primers flanking the deleted region

• Next-generation sequencing of amplicons to identify smaller insertions/deletions

Protein-level validation:

• Western blot analysis using multiple antibodies recognizing different epitopes:

  • N-terminal antibodies (detecting Synpo-S and Synpo-L)

  • C-terminal antibodies (detecting only Synpo-L and Synpo-T)

  • Internal region antibodies (detecting Synpo-S and Synpo-L)

Functional assessment:

• Evaluate baseline phenotypes in podocytes and telencephalic neurons

• Challenge knockout models with injury models to assess protective role

• Examine actin reorganization and focal adhesion dynamics

A comprehensive validation approach ensures the knockout model's reliability for subsequent functional studies investigating SYNPO's role in podocyte homeostasis and neuronal function.

What are the critical considerations for immunofluorescence localization of SYNPO in neuronal and kidney tissues?

Accurate immunofluorescence localization of SYNPO requires careful attention to tissue-specific protocols:

Neuronal tissue considerations:

• SYNPO localizes to a subset of exclusively telencephalic synapses

• Expression is differentiation-dependent during postnatal maturation

• In cultured hippocampal neurons, expression follows differentiation patterns

• Use neuronal markers (e.g., PSD-95) for colocalization studies to identify synaptic structures

Kidney tissue considerations:

• SYNPO localizes to differentiated podocytes (glomerular visceral epithelial cells)

• Co-localization with alpha-actinin is expected

• Weaker additional reaction with arterial endothelial cells may occur

• SYNPO does not react with parietal cells, which can serve as negative controls

Optimized immunofluorescence protocol:

• Fixation and permeabilization as described in FAQ 2.1

• Block with serum from the species in which the secondary antibody was raised (30 min)

• Incubate with primary antibody for 1 hour at room temperature in a moist chamber

• Wash 3× with PBS

• Incubate with appropriate fluorescent secondary antibody for 30-60 minutes at room temperature

• Wash 3× with PBS

• Briefly immerse in ethanol

• Air dry and cover with mounting medium

Cross-validation with multiple antibodies recognizing different SYNPO epitopes strengthens the reliability of localization data.

How can I troubleshoot contradictory results when studying SYNPO in disease models?

When facing contradictory results in SYNPO research, consider these methodological troubleshooting approaches:

Antibody-related factors:

• Epitope masking: Different fixation protocols may mask specific epitopes

• Isoform specificity: Ensure antibodies detect the relevant isoforms for your tissue

• Cross-reactivity: Validate antibody specificity with appropriate controls

• Lot-to-lot variation: Consider using monoclonal antibodies for consistent results

Sample preparation issues:

• Protein degradation: The 44-45 kDa band often represents degraded SYNPO; use fresh samples and protease inhibitors

• Post-translational modifications: Phosphorylation status may affect antibody recognition

• Extraction methods: Different buffers may yield variable protein extraction efficiency

Experimental design considerations:

• Tissue-specific expression: Neural (100 kDa) vs. renal (110 kDa) isoforms

• Developmental timing: Expression changes during postnatal maturation

• Disease state influence: SYNPO expression may be altered in pathological conditions

• Species differences: Confirm antibody reactivity with your experimental species

Validation strategies:

• Multiple antibodies: Use antibodies recognizing different epitopes

• Multiple techniques: Combine IF, WB, and IHC for comprehensive analysis

• Genetic models: Include SYNPO knockout controls when possible

• Positive controls: Include tissues known to express SYNPO (brain, kidney)

When reporting contradictory results, document all methodological details, including fixation, antibody information, and experimental conditions to facilitate replication and resolution of discrepancies.