CASK Antibody

What is CASK protein and what structural domains are significant for antibody targeting?

CASK is a multidomain scaffolding protein that belongs to the MAGUK family. It contains several functional domains including a CaMK domain, two L27 domains, a PDZ domain, an SH3 domain, and a guanylate kinase domain from N- to C-terminal . Most commercial antibodies target specific regions within these domains, with many recognizing epitopes in the CaMK domain (aa 300-500) or regions between aa 318-415 . When selecting antibodies for experimental applications, considering the specific domain you're investigating is critical for obtaining reliable results.

What are the common applications for CASK antibodies in neurobiological research?

CASK antibodies are widely employed in multiple applications including Western Blot (1:500-1:1000 dilution), Immunocytochemistry/Immunofluorescence (1:50-1:200), Immunohistochemistry-Paraffin (1:100-1:200), and Flow Cytometry (1:50-1:100) . For studying neuronal systems, these antibodies can identify CASK localization at both presynaptic terminals (where it interacts with Neurexin) and postsynaptic regions, making them valuable tools for examining synaptic architecture and function . When designing experiments, it's essential to validate antibody specificity in your experimental system through appropriate controls.

What species reactivity should be considered when selecting CASK antibodies?

Current commercially available CASK antibodies demonstrate varying species reactivity profiles. Many antibodies show reactivity against human and rat CASK proteins . Some antibodies recognize additional species such as mouse (O70589), chicken, xenopus, and zebrafish . When planning cross-species studies, it's crucial to select antibodies with confirmed reactivity against your species of interest and to validate this reactivity through preliminary experiments, as sequence variations between species may affect epitope recognition.

How can CASK antibodies be utilized to investigate MICPCH syndrome mechanisms?

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is associated with CASK gene mutations. CASK antibodies have been instrumental in characterizing how various mutations affect protein expression and function. In knockout mouse models replicating MICPCH, researchers have used CASK antibodies to verify complete protein loss and study the downstream effects on cerebellar granule cells . For investigating the functional significance of specific mutations, antibodies can be paired with rescue experiments where wild-type or mutant CASK is reintroduced into knockout systems. This approach has helped identify that the CaMK, PDZ, and SH3 domains are essential for cerebellar granule cell survival, while the L27 and guanylate kinase domains appear dispensable for this function .

What methodological considerations are important when using CASK antibodies to study protein-protein interactions?

CASK interacts with multiple proteins through its various domains, making antibody selection critical for interaction studies. The CaMK domain interacts with Mint1, Caskin1, Tiam1, and Liprin-α proteins, often in competitive binding relationships . When designing co-immunoprecipitation experiments, consider whether your antibody's epitope overlaps with protein binding sites, as this could interfere with detecting certain interactions. Recent structural analysis using machine learning tools like AlphaFold 2.2 has revealed that patient-derived mutations in the CaMK domain disrupt the CASK-Liprin-α2 binding interface, highlighting how antibodies recognizing this region can be particularly useful for studying pathological mechanisms . For optimal results, use antibodies targeting domains not involved in the specific interaction you're studying.

How can CASK antibodies be applied in CRISPR/Cas9 gene editing experiments?

CRISPR/Cas9 gene editing has enabled the creation of isogenic CASK knockout cell lines, providing valuable models for studying CASK function without the confounding variables of different genetic backgrounds . CASK antibodies are essential tools for validating successful gene editing. In human embryonic stem cell (hESC) models, researchers have used immunostaining with anti-CASK antibodies to confirm protein loss in cells targeted for CASK deletion . When designing similar experiments, it's advisable to use antibodies recognizing epitopes in exons targeted by your CRISPR guide RNAs to ensure complete knockout validation. Additionally, these antibodies can be used to quantify protein levels in heterozygous models or to detect truncated proteins produced by frameshift mutations.

What are the optimal protocols for using CASK antibodies in Western blot applications?

For Western blot applications, CASK antibodies are typically used at dilutions ranging from 1:100 to 1:1000 . CASK protein has a predicted molecular weight of approximately 59-100 kDa (theoretical MW of 59 kDa, but often observed at higher weights due to post-translational modifications) . To optimize Western blot protocols:

• Sample preparation: Cell or tissue lysates should be prepared in RIPA or similar buffers containing protease inhibitors

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Transfer conditions: Standard semi-dry or wet transfer to PVDF membranes

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Overnight at 4°C in blocking buffer

• Detection: HRP-conjugated secondary antibodies followed by ECL detection

It's important to note that "the observed molecular weight of the protein may vary from the listed predicted molecular weight due to post-translational modifications, post-translation cleavages, relative charges, and other experimental factors" .

What controls should be incorporated when using CASK antibodies in immunohistochemistry?

When using CASK antibodies for immunohistochemistry or immunocytochemistry, several controls are essential:

• Positive control tissues: Human placenta tissue has been validated for CASK immunohistochemistry

• Negative controls: Omission of primary antibody and use of isotype-matched irrelevant antibodies

• Knockout/knockdown controls: When available, CASK knockout or knockdown samples provide the gold standard for antibody specificity validation

• Competing peptide controls: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

For immunohistochemistry-paraffin, researchers typically use dilutions of 1:100-1:200, with antigen retrieval methods optimized for the specific tissue type . For cultured cells, fixation with 4% PFA/4% sucrose in PBS has been successful for CASK immunostaining .

What strategies can resolve inconsistent results between different CASK antibodies?

Discrepancies between different CASK antibodies may arise from:

• Epitope differences: Antibodies targeting different domains may produce varying results, especially if certain domains are affected by experimental conditions or mutations

• Antibody specificity: Some antibodies may recognize non-specific proteins in addition to CASK

• Sample preparation differences: Different fixation methods or buffer compositions can affect epitope accessibility

To resolve inconsistencies:

• Compare antibodies targeting different domains of CASK

• Validate results using genetic approaches (siRNA knockdown or CRISPR knockout)

• Consider use of both monoclonal and polyclonal antibodies for confirmation

• Perform peptide competition assays to verify specificity

• Use multiple detection methods (e.g., both Western blot and immunostaining)

How should experiments be designed to study CASK mutations using antibodies?

When studying CASK mutations, careful experimental design is critical. Based on recent research examining MICPCH syndrome-associated mutations:

• Model selection: Both in vitro (cultured cells) and in vivo (animal) models provide complementary information. Female heterozygous CASK KO mice replicate progressive cerebellar hypoplasia observed in MICPCH syndrome

• Rescue experiments: These are powerful for determining functional significance of specific domains or mutations

  • Culture cerebellar granule (CG) cells from CASK KO mice

  • Infect with lentivirus expressing wild-type or mutant CASK

  • Assess cellular survival and function using appropriate assays

  • Use antibodies to confirm expression of rescue constructs

• Domain-specific analysis: Test multiple domain deletion mutants to identify critical functional regions

  • The CaMK, PDZ, and SH3 domains have been identified as necessary for CG cell survival

  • The L27 and guanylate kinase domains appear dispensable for this function

• Patient mutation analysis: Introduce specific patient-derived mutations into expression constructs

  • Test functional rescue capability

  • Use structural prediction tools (e.g., AlphaFold) to correlate structural changes with functional deficits

  • Apply antibodies to assess protein expression levels and localization

What are the considerations for quantifying CASK expression levels in research samples?

Accurate quantification of CASK expression requires careful methodological considerations:

• Normalization strategy:

  • For Western blot: Normalize to housekeeping proteins like tubulin, actin, or GAPDH

  • For immunofluorescence: Co-stain with neuronal markers (e.g., tubulin3/Tuji1) and normalize signal to cell number or area

• Sample preparation:

  • Tissue samples: Consider regional variability in CASK expression

  • Cell culture: Standardize cell density and culture conditions

• Antibody selection:

  • Choose antibodies with proven quantitative linearity

  • Consider using multiple antibodies targeting different domains for validation

• Data analysis:

  • Apply appropriate statistical methods based on sample distribution

  • Present data normalized to controls rather than absolute values

  • Report biological replicates from independent experiments rather than technical replicates

How can CASK antibodies be utilized to analyze subcellular localization changes?

CASK demonstrates dual localization to the plasma membrane and nucleus , and its distribution may change under various conditions or in disease states. Strategies for analyzing subcellular localization include:

• Subcellular fractionation followed by Western blot:

  • Separate nuclear, cytoplasmic, and membrane fractions using differential centrifugation

  • Analyze CASK distribution across fractions using Western blot

  • Include fraction-specific markers (e.g., Histone H3 for nuclear, Na+/K+ ATPase for membrane)

• High-resolution immunofluorescence:

  • Use confocal or super-resolution microscopy for precise localization

  • Employ co-staining with compartment-specific markers (membrane markers, nuclear stains)

  • Quantify colocalization using appropriate software and statistical measures

• Proximity ligation assays:

  • Detect interactions with known binding partners in specific subcellular compartments

  • Use antibodies against CASK and its binding partners (e.g., Neurexin, Liprin-α, Mint1)

• Live cell imaging:

  • For dynamic studies, use fluorescent protein-tagged CASK constructs

  • Validate localization patterns using antibody staining of fixed cells

What are common technical challenges when working with CASK antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with CASK antibodies:

• Background signal:

  • Increase blocking time/concentration (use 5% BSA or milk)

  • Optimize antibody dilution through titration experiments

  • Try different secondary antibodies or detection systems

  • Include additional washing steps with higher detergent concentration

• Weak or absent signal:

  • Verify sample preparation (protein denaturation for Western blot, fixation conditions for immunostaining)

  • Modify antigen retrieval methods for tissue sections

  • Try alternative antibodies targeting different epitopes

  • Check antibody storage conditions and expiration

• Multiple bands in Western blot:

  • Determine if bands represent isoforms, post-translational modifications, or degradation products

  • Use positive controls with known molecular weight

  • Include peptide competition controls to identify specific bands

• Variability between experiments:

  • Standardize all protocols in detail

  • Prepare larger batches of antibody dilutions

  • Include internal controls in each experiment

  • Maintain consistent imaging parameters

How can researchers validate CASK antibody specificity?

Antibody validation is critical for ensuring reliable results. Multiple approaches should be combined:

• Genetic validation:

  • Use CASK knockout or knockdown samples as negative controls

  • Wildtype vs. CASK KO cerebellar granule cells show clear differential staining when properly validated

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • This should abolish specific staining/bands

• Multiple antibody concordance:

  • Use multiple antibodies targeting different epitopes

  • Consistent results across antibodies increase confidence

• Recombinant expression:

  • Overexpress tagged CASK constructs

  • Confirm antibody detection of overexpressed protein

• Mass spectrometry:

  • Immunoprecipitate CASK and confirm identity by mass spectrometry

  • This is particularly important for validating new antibodies

What storage and handling practices optimize CASK antibody performance?

Proper antibody storage and handling significantly impact experimental outcomes:

• Storage conditions:

  • Store lyophilized antibodies at 4°C until reconstitution

  • After reconstitution, aliquot and store at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles by making single-use aliquots

  • Do not freeze reconstituted antibodies before aliquoting

• Reconstitution:

  • Add 50 μl H₂O to get a 1mg/ml solution in PBS (for typical lyophilized preparations)

  • Allow complete dissolution before aliquoting

• Working dilutions:

  • Prepare fresh working dilutions for each experiment

  • Use high-quality diluents with appropriate preservatives

  • Return stock solutions to proper storage promptly

• Quality control:

  • Test new lots against previous lots before use in critical experiments

  • Include positive control samples in each experiment

  • Monitor signal-to-noise ratio over time to detect potential antibody degradation