CSC1 Antibody

What is CSC1 and what are its primary functions?

CSC1 (TMEM63C) functions as an osmosensitive cation channel that is preferentially activated upon hypotonic stress. It is enriched in mitochondria-ER contact sites where it regulates metabolite flux and organelle morphologies in response to osmotic changes. Research has shown that CSC1 plays a crucial role in regulating mitochondrial motility and function in motor neuron axons. Additionally, it is required for maintaining the functional integrity of the kidney glomerular filtration barrier . These diverse functions make CSC1 an important target for studies related to cell volume regulation, mitochondrial dynamics, and kidney physiology.

Where is CSC1 primarily expressed in mammalian tissues?

CSC1/TMEM63C exhibits tissue-specific expression patterns across mammalian species. It is prominently expressed in kidney tissues, particularly in podocytes, where it appears to play a critical role in maintaining glomerular filtration barrier integrity. Notably, patients with focal segmental glomerulosclerosis show specific TMEM63C loss in podocytes . The protein is also found in neural tissues, as evidenced by detection in mouse cerebral cortex tissue lysates in western blot experiments . This expression pattern aligns with CSC1's functional involvement in both renal and neuronal physiology.

What types of CSC1 antibodies are available for research applications?

Currently, researchers have access to several types of CSC1/TMEM63C antibodies for experimental applications:

These antibodies offer complementary research tools, with the monoclonal providing high specificity and the polyclonal potentially offering enhanced signal detection across multiple epitopes .

How can I validate the specificity of a CSC1 antibody for my research?

Validating CSC1 antibody specificity requires a multi-faceted approach:

• Positive and negative control samples: Use tissues/cells known to express CSC1 (such as kidney tissue or cerebral cortex) as positive controls, and consider using CSC1-knockout samples or tissues with minimal expression as negative controls.

• Expected molecular weight verification: Confirm that the detected band appears at the expected 93 kDa in western blot applications.

• Cross-reactivity assessment: Test the antibody against related proteins or in species not listed in the reactivity profile to ensure specificity.

• Consistency across applications: If using the antibody for multiple techniques (WB, ICC/IF, etc.), verify consistent detection patterns across platforms.

• Literature comparison: Compare your results with published data on CSC1 localization and expression patterns.

This systematic validation approach ensures reliable results in subsequent experiments .

Why do some CSC1 antibodies show inconsistent results across species?

Antibody cross-reactivity across species depends on epitope conservation between orthologs. Based on research with other proteins like Pneumocystis carinii, we know that even closely related species can exhibit significant antigenic differences . For CSC1/TMEM63C specifically, antibody performance may vary between human, mouse, and rat samples due to:

Most commercial CSC1 antibodies are validated for specific species reactivity, with some showing broader cross-reactivity than others. When working with non-validated species, researchers should perform careful preliminary validation .

What are the recommended protocols for using CSC1 antibodies in Western blot applications?

For optimal Western blot results with CSC1/TMEM63C antibodies, follow these specialized protocols:

• Sample preparation:

  • Do not boil samples prior to loading to avoid formation of high molecular weight CSC1 aggregates

  • Use standard cell/tissue lysis buffers containing protease inhibitors

• Dilution ratios:

  • For monoclonal antibodies (e.g., EPR19814): Use at 1/1000 dilution

  • For polyclonal antibodies (e.g., 23115-1-AP): Use at 1/500-1/2000 dilution

• Blocking conditions:

  • Block membranes with 5% non-fat dry milk (NFDM) in TBST

• Detection optimization:

  • Exposure times may need adjustment based on tissue type (e.g., 3 minutes for mouse cerebral cortex, 10 seconds for other tissues)

  • For weaker signals, consider longer antibody incubation times at 4°C overnight

Following these protocols will help minimize background and maximize specific CSC1 detection .

How should I prepare samples for immunofluorescence detection of CSC1?

For successful immunofluorescence detection of CSC1/TMEM63C, follow these methodological recommendations:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature, as overfixation may mask the CSC1 epitope.

• Permeabilization: A gentle permeabilization with 0.1-0.2% Triton X-100 for 5-10 minutes is recommended to preserve membrane structure while allowing antibody access.

• Blocking: Block with 5% normal serum (matching the species of the secondary antibody) for 1 hour to reduce background.

• Antibody dilution: For primary CSC1 antibodies, start with manufacturers' recommended dilutions (typically 1:100-1:500) in blocking buffer.

• Counterstaining: Consider co-staining with mitochondrial or ER markers to visualize CSC1 enrichment at mitochondria-ER contact sites.

• Microscopy settings: Use confocal microscopy for optimal visualization of subcellular localization patterns .

What considerations are important when performing immunoprecipitation with CSC1 antibodies?

When immunoprecipitating CSC1/TMEM63C, several technical considerations can improve success:

• Lysis conditions: Use mild, non-denaturing lysis buffers to preserve protein interactions and native conformation.

• Pre-clearing lysates: Pre-clear your lysates with protein A/G beads to reduce non-specific binding.

• Antibody selection: Choose antibodies specifically validated for IP applications, such as the rabbit recombinant monoclonal EPR19814 .

• Incubation conditions: Allow sufficient time (4-16 hours at 4°C) for antibody-protein binding.

• Washing stringency: Balance between removing non-specific interactions and preserving specific ones through optimized wash buffer composition.

• Elution strategy: Consider native elution methods if planning to assess CSC1 channel function post-IP.

• Controls: Always include an isotype control antibody IP to identify non-specific interactions.

These considerations help ensure specific recovery of CSC1 and its interacting partners .

Why does CSC1 form high molecular weight aggregates in western blot applications?

CSC1/TMEM63C has a tendency to form high molecular weight aggregates during sample preparation for western blot, particularly when samples are boiled. This aggregation occurs due to several factors:

• Transmembrane domain aggregation: As a multi-pass membrane protein with several transmembrane domains, CSC1 can form aggregates when these hydrophobic regions interact during denaturation.

• Heat-induced conformational changes: Boiling can cause irreversible protein misfolding and subsequent aggregation.

• Cysteine-mediated cross-linking: Heat may promote disulfide bond formation between CSC1 molecules.

To avoid this issue, manufacturers specifically recommend not boiling samples in sample buffer prior to western blot testing. Instead, heat samples to a lower temperature (37°C for 10 minutes) to promote partial denaturation without aggregation . This modification is crucial for detecting the proper 93 kDa band rather than high molecular weight smears.

What strategies can improve detection sensitivity for low CSC1 expression samples?

When working with samples expressing low levels of CSC1/TMEM63C, these strategies can enhance detection sensitivity:

• Sample enrichment: Consider subcellular fractionation to isolate membrane fractions where CSC1 is localized.

• Signal amplification systems: Use highly sensitive ECL substrates for western blot or tyramide signal amplification for immunostaining.

• Antibody optimization: Test different antibody concentrations and extended incubation times (4°C overnight).

• Increased protein loading: Load more total protein (50-100 μg) for western blot applications.

• Reduced washing stringency: Use gentler washing conditions to preserve antibody-antigen interactions.

• Alternative detection methods: Consider using more sensitive detection methods like proximity ligation assay (PLA) for very low abundance targets.

• Extended exposure times: For western blot, using longer exposure times may be necessary (as noted in protocols where mouse cerebral cortex required 3-minute exposures compared to 10 seconds for other tissues) .

How can I reduce background when using CSC1 antibodies in immunocytochemistry?

High background is a common challenge when using CSC1/TMEM63C antibodies for immunocytochemistry. These steps can significantly improve signal-to-noise ratio:

• Optimize fixation protocol: Over-fixation can increase background; test shorter fixation times.

• Enhance blocking: Consider extended blocking (2+ hours) with 5-10% normal serum from the same species as the secondary antibody.

• Include protein blockers: Add 0.1-0.5% BSA to antibody dilution buffers to reduce non-specific binding.

• Pre-adsorb secondary antibodies: Incubate secondary antibodies with fixed, permeabilized cells lacking the target to remove cross-reactive antibodies.

• Increase washing steps: Add additional, longer washes between antibody incubations.

• Reduce antibody concentrations: Titrate primary and secondary antibody concentrations to find the optimal signal-to-noise ratio.

• Use fluorophores with minimal spectral overlap: Choose fluorophores that minimize autofluorescence interference from the sample.

These approaches can dramatically improve CSC1 immunostaining quality and specificity .

How can CSC1 antibodies be used to study mitochondria-ER contact sites?

CSC1/TMEM63C is enriched at mitochondria-ER contact sites, making its antibodies valuable tools for studying these crucial cellular junctions. Advanced research applications include:

• Super-resolution microscopy: Using CSC1 antibodies in conjunction with STED or STORM microscopy can visualize the precise spatial arrangement of CSC1 at contact sites with nanometer resolution.

• Proximity ligation assays (PLA): Combining CSC1 antibodies with antibodies against known ER or mitochondrial markers in PLA experiments can quantitatively assess changes in organelle proximity under different physiological conditions.

• Immunoelectron microscopy: Gold-labeled CSC1 antibodies can reveal ultrastructural details of CSC1 localization at contact sites.

• Co-immunoprecipitation studies: CSC1 antibodies can help identify novel protein complexes at mitochondria-ER junctions when coupled with mass spectrometry analysis.

• Live-cell imaging: When combined with other fluorescently tagged organelle markers, immunofluorescence with CSC1 antibodies can track dynamic changes in contact site formation during cellular responses to osmotic stress .

What role does CSC1 play in kidney pathophysiology and how can antibodies help study this?

CSC1/TMEM63C's role in kidney pathophysiology, particularly in podocyte function and glomerular filtration, can be investigated using antibodies through several advanced approaches:

• Immunohistochemical analysis of patient biopsies: CSC1 antibodies can be used to assess protein expression patterns in kidney diseases, particularly focal segmental glomerulosclerosis where TMEM63C loss in podocytes has been observed .

• Correlative microscopy: Combining CSC1 immunostaining with functional assays of glomerular filtration barrier integrity can reveal structure-function relationships.

• In vitro podocyte models: CSC1 antibodies can track protein localization and abundance in cultured podocytes under disease-mimicking conditions.

• Phosphorylation status assessment: Phospho-specific antibodies (when developed) could reveal CSC1 activation states during kidney injury.

• Therapeutic target validation: Antibodies can assess whether candidate compounds restore CSC1 expression or localization in disease models.

These applications provide valuable insights into CSC1's contribution to kidney disease pathogenesis and potential therapeutic approaches .

How can researchers study the relationship between CSC1 and osmotic stress response pathways?

To investigate CSC1/TMEM63C's role in osmotic stress response pathways, researchers can employ antibodies in these advanced experimental designs:

• Time-course immunoblotting: Use CSC1 antibodies to track protein expression, modification, or degradation following hypotonic challenges at various timepoints.

• Proximity-dependent biotinylation (BioID): Combine with CSC1 antibodies to identify proteins that interact with CSC1 specifically under osmotic stress conditions.

• Calcium imaging correlation: Pair calcium imaging during osmotic stress with subsequent CSC1 immunostaining to correlate channel localization with functional calcium signaling.

• CRISPR-edited CSC1 variants: Use antibodies to verify expression of engineered CSC1 mutants designed to test channel properties or interaction domains.

• Tissue-specific expression analysis: Apply CSC1 antibodies to examine differential responses to osmotic challenges across tissue types.

• Subcellular fractionation studies: Track CSC1 redistribution between membrane compartments during adaptation to osmotic stress.

These approaches can reveal mechanistic insights into how CSC1 contributes to cellular osmosensing and downstream signaling events .

How should researchers interpret differences in CSC1 banding patterns across experimental conditions?

Interpreting variations in CSC1/TMEM63C banding patterns requires careful analysis of several factors:

• Multiple bands vs. single band: A single clear band at 93 kDa indicates detection of monomeric CSC1, while multiple bands may represent:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Proteolytic processing

  • Alternative splicing isoforms

  • Sample preparation artifacts (particularly high molecular weight bands from aggregation)

• Band intensity changes: When comparing conditions, consider whether changes reflect:

  • Altered protein expression

  • Modified subcellular localization affecting extraction efficiency

  • Changes in protein stability or turnover

• Band migration shifts: Slight changes in apparent molecular weight may indicate:

  • Post-translational modifications

  • Conformational changes affecting SDS binding

  • Buffer composition effects

• Tissue-specific patterns: Different tissues may show distinct banding patterns due to tissue-specific processing or modifications

Remember that avoiding sample boiling is critical for proper CSC1 detection, as heat can cause the formation of high molecular weight aggregates that appear as additional bands or smears in the gel .

What controls should be included when publishing research using CSC1 antibodies?

For scientifically rigorous publication of research involving CSC1/TMEM63C antibodies, include these essential controls:

• Positive and negative tissue controls: Include samples known to express (kidney, brain) or lack CSC1.

• Knockdown/knockout validation: When possible, include CSC1 knockdown/knockout samples to demonstrate antibody specificity.

• Loading controls: For western blot, include appropriate loading controls (β-actin, GAPDH) and quantitative normalization.

• Secondary-only controls: For immunostaining, include samples treated with only secondary antibody to assess background.

• Non-specific IgG controls: For immunoprecipitation, include isotype-matched non-specific IgG.

• Concentration-matched controls: When comparing effects of treatments on CSC1 levels, ensure equal protein loading.

• Reproducibility evidence: Demonstrate consistent results across multiple experimental replicates.

• Antibody validation documentation: Include catalog numbers, lot numbers, and validation performed (especially for antibodies without extensive citation history).

These controls ensure data reliability and facilitate peer reviewer confidence in your findings .

How can quantitative analysis of CSC1 expression be standardized across research groups?

To enhance reproducibility and enable cross-laboratory comparisons of CSC1/TMEM63C expression data, researchers should implement these standardization practices:

• Standardized reference samples: Establish common positive control lysates/tissues that can be shared between laboratories.

• Absolute quantification methods: Consider using recombinant CSC1 protein standards of known concentration to generate standard curves.

• Consistent normalization strategy: Adopt uniform loading controls and normalization procedures (e.g., normalizing to total protein via stain-free technology rather than variable housekeeping proteins).

• Detailed protocol sharing: Document and share complete protocols including sample preparation, antibody dilutions, incubation times, and image acquisition settings.

• Digital image analysis standards: Use consistent thresholding and quantification parameters for densitometry or immunofluorescence intensity measurements.

• Multi-antibody validation: When possible, confirm findings using multiple antibodies targeting different CSC1 epitopes.

• Method cross-validation: Validate protein expression changes across multiple techniques (e.g., western blot, qPCR, mass spectrometry).