CA4 Antibody

What tissues express CA4 and can serve as positive controls?

CA4 demonstrates specific expression patterns across multiple tissues, making certain samples ideal as positive controls. Human CA4 is prominently expressed in lung, kidney, brain (cerebellum), and colon tissues . In human colon, CA4 localizes specifically to membranes of epithelial cells in mucosal glands . In human kidney, CA4 expression is concentrated in renal tubules . Mouse CA4 shows strong expression in kidney tissues, particularly in the cytoplasm of epithelial cells, and is also detectable in lung tissue . When designing experiments, these tissue types should be incorporated as positive controls to validate antibody performance.

What are the optimal dilutions for CA4 antibodies in different applications?

Optimal dilutions vary by application technique and must be empirically determined:

Always validate these dilutions in your experimental system, as optimal concentrations may vary based on sample type, fixation method, and detection system .

What molecular weights should be expected when detecting CA4?

Interestingly, CA4 detection reveals different apparent molecular weights depending on the experimental system:

These variations likely reflect tissue-specific post-translational modifications, different experimental conditions, or detection methods . When analyzing your results, consider these reference points but expect some variation across experimental conditions.

How should CA4 antibodies be stored to maintain activity?

Proper storage is critical for antibody stability. For short-term storage (up to one month), CA4 antibodies can typically be stored at 2-8°C without detectable loss of activity . For long-term storage, aliquot the antibody and store at -20°C to avoid repeated freeze-thaw cycles that can compromise activity . According to manufacturer guidelines, antibody products maintain stability for twelve months from the date of receipt when stored properly . Always follow specific storage instructions provided by the manufacturer for the particular antibody you're using.

How can CA4 antibodies help investigate the role of CA4 in pH regulation?

CA4's role in catalyzing the reversible hydration of carbon dioxide (CO₂ + H₂O = HCO₃⁻ + H⁺) makes it central to pH regulation in multiple physiological contexts . Researchers can employ CA4 antibodies in co-localization studies with pH-sensitive probes to visualize spatial relationships between CA4 expression and local pH environments. In nasal epithelial studies, carbonic anhydrase IV has been shown to influence ciliary motility through pH regulation mechanisms .

For mechanistic investigations, researchers can combine antibody-based detection of CA4 with functional assays measuring pH changes or bicarbonate transport. This approach is particularly valuable when studying CA4's functions in renal tubular acidification, where proper pH balance is crucial for kidney function . Additionally, using CA4 antibodies in conjunction with inhibitors of carbonic anhydrase activity can help delineate specific contributions of CA4 to physiological pH regulation compared to other carbonic anhydrase isoforms.

What is the significance of CA4 mutations in retinitis pigmentosa research?

CA4 has direct clinical relevance to retinitis pigmentosa (RP) research, as a specific genetic mutation (Arg14 to Trp in the signal peptide) of CA4 has been found to cosegregate with the RP17 form of retinitis pigmentosa . This mutation was identified in two large families with the condition and was absent in 36 unaffected family members and 100 controls .

Researchers investigating this connection can employ CA4 antibodies to:

• Compare wild-type and mutant CA4 localization in retinal tissue samples

• Evaluate how the mutation affects CA4 protein expression, stability, and function

• Develop model systems to study pathogenic mechanisms

• Screen potential therapeutic interventions targeting CA4-related pathways

The specific relationship between CA4 and retinitis pigmentosa highlights the importance of this enzyme beyond its canonical role in acid-base balance, suggesting broader implications in neurosensory physiology .

How does the GPI-anchored nature of CA4 affect experimental approaches?

CA4 is a GPI-anchored membrane enzyme expressed on the luminal surfaces of pulmonary capillaries and proximal renal tubules . This unique membrane attachment has several important implications for experimental design:

• Subcellular fractionation: Standard protocols may need modification to efficiently extract GPI-anchored proteins, which partition into detergent-resistant membrane microdomains.

• Tissue fixation and processing: Harsh fixation may disrupt GPI anchors, potentially leading to artifactual localization patterns. Cross-validation using multiple fixation approaches is recommended.

• Epitope accessibility: The membrane orientation of CA4 may restrict antibody access to certain epitopes, particularly in intact cell applications like flow cytometry.

• Functional studies: When investigating CA4 activity, researchers should consider its restricted orientation at the membrane, which may limit substrate accessibility to one side of the membrane.

Experiments designed to study CA4 should incorporate appropriate controls and methodologies that account for these unique properties of GPI-anchored proteins .

What are optimal conditions for immunohistochemical detection of CA4?

Successful immunohistochemical detection of CA4 requires careful optimization of several parameters. Based on validated protocols from the literature, researchers should consider:

For paraffin-embedded sections:

• Heat-induced epitope retrieval using basic pH buffer (such as VisUCyte Antigen Retrieval Reagent-Basic) is recommended prior to primary antibody incubation

• Optimal antibody concentrations range from 1:100-1:500 dilution or approximately 3 μg/mL

• Incubation times of 1 hour at room temperature or overnight at 4°C are commonly effective

• HRP-DAB detection systems provide good visualization of CA4 in tissue sections

• Hematoxylin counterstaining helps visualize tissue architecture alongside CA4 expression

For frozen sections:

• In mouse tissues, perfusion fixation followed by freezing has been successfully employed

• Overnight incubation at 4°C with anti-CA4 antibody (3 μg/mL) shows good results

• Anti-Goat HRP-DAB detection systems are effective for visualizing CA4 in frozen sections

These conditions should be further optimized for specific experimental needs, particularly when working with different species or tissue types .

How can researchers differentiate between various carbonic anhydrase isoforms?

Distinguishing between different carbonic anhydrase isoforms requires careful selection of antibodies and experimental controls:

• Antibody selection: Choose antibodies raised against unique epitopes of CA4 that have minimal sequence homology with other CA isoforms. Review cross-reactivity data provided by manufacturers.

• Tissue selection: Leverage the distinct tissue expression patterns of different CA isoforms. CA4 is predominantly expressed in renal tubules, pulmonary capillaries, and intestinal epithelium .

• Co-labeling approaches: Perform dual immunostaining with antibodies against CA4 and other CA isoforms to identify regions of exclusive or overlapping expression.

• Molecular weight verification: Use Western blot to confirm the molecular weight of the detected protein matches the expected size for CA4 (approximately 33-40 kDa for human, 35-52 kDa for mouse) .

• Knockout/knockdown controls: When available, use tissues or cells with confirmed CA4 knockout/knockdown as negative controls to validate antibody specificity.

This multilayered approach helps ensure that the detected signal specifically represents CA4 rather than other carbonic anhydrase family members.

What experimental controls are essential when using CA4 antibodies?

Rigorous experimental controls are critical for generating reliable data with CA4 antibodies:

Additionally, when interpreting Western blot data, researchers should verify that the detected bands correspond to the expected molecular weight of CA4 (with consideration for splice variants and post-translational modifications) as demonstrated in the research literature .

Why might CA4 appear at different molecular weights in different detection systems?

The observed molecular weight variations for CA4 across different experimental systems stem from several factors:

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications may differ between tissues or cell types, affecting protein migration.

• Detection system differences: Simple Western™ consistently shows higher apparent molecular weights (40 kDa for human, 51-52 kDa for mouse) compared to traditional Western blot (33 kDa for human, 35 kDa for mouse) . This may reflect differences in protein denaturation, separation matrices, or detection chemistry.

• GPI anchor retention: The presence or absence of the GPI anchor can affect apparent molecular weight.

• Species differences: Human CA4 (33-40 kDa) and mouse CA4 (35-52 kDa) show different molecular weight profiles, potentially reflecting evolutionary differences in protein structure or post-translational processing .

• Tissue-specific processing: Evidence suggests tissue-specific processing of CA4, as demonstrated by the different molecular weights observed in different tissues .

When interpreting CA4 molecular weight data, researchers should consider these factors and include appropriate molecular weight markers and controls.

How can researchers troubleshoot weak or absent CA4 signal in immunohistochemistry?

When faced with weak or absent CA4 signal in immunohistochemistry, consider these systematic troubleshooting approaches:

• Epitope masking: CA4's GPI anchor may cause epitope masking. Try alternative epitope retrieval methods:

  • Increase heat-induced epitope retrieval time or temperature

  • Test different pH buffers for antigen retrieval

  • Consider enzymatic retrieval methods

• Antibody concentration: Titrate antibody concentration more precisely. For paraffin sections, the recommended range of 1:100-1:500 is broad; narrow this range with sequential dilutions .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C if room temperature incubation yields weak signals .

• Detection system sensitivity: Switch to a more sensitive detection system. If using a standard ABC method, consider switching to polymer-based detection systems like VisUCyte™ HRP Polymer .

• Tissue fixation issues: Overfixation can mask epitopes. Test antibody on frozen sections if paraffin sections consistently show poor results .

• Sample storage artifacts: Prolonged storage of cut sections can reduce antigenicity. Use freshly cut sections from well-preserved blocks.

• Expression levels: Confirm CA4 expression in your specific tissue/cell type through literature or database searches, as expression levels vary significantly between tissues .

Systematic application of these approaches while changing one variable at a time will help identify the source of the problem.

How should researchers interpret CA4 staining patterns in different tissues?

The interpretation of CA4 staining patterns requires careful consideration of tissue-specific expression patterns and subcellular localization:

In Human Tissues:

• Colon: CA4 primarily localizes to membranes of epithelial cells in mucosal glands . Cytoplasmic staining in these cells should be minimal, and strong membrane positivity indicates specific staining.

• Kidney: Expect strong positivity in renal tubules . The staining should be more intense in proximal tubules compared to distal segments.

• Lung: CA4 localizes specifically to pulmonary capillaries . The staining pattern should appear as a fine network corresponding to the capillary bed structure.

In Mouse Tissues:

• Kidney: CA4 shows strong expression in the cytoplasm of epithelial cells . This differs from human kidney, where membrane localization may be more prominent.

• Lung: Similar to human lung, expect capillary staining, though the pattern may differ slightly due to species-specific vascular architecture .

• Immune cells: CA4 expression has been detected on IL-5-activated murine eosinophils, suggesting a role in immune function .

When interpreting staining patterns, always compare with positive control tissues and refer to published patterns. Unexpected staining patterns should be validated with alternative detection methods or antibodies to rule out non-specific binding .

How can CA4 antibodies be applied in respiratory and renal physiology research?

CA4 antibodies offer valuable tools for investigating fundamental aspects of respiratory and renal physiology:

Respiratory Applications:

• Mapping CA4 distribution across pulmonary capillary networks to understand regional variations in CO₂ exchange capacity

• Investigating CA4's role as the principal CO₂ taste sensor, which has implications for respiratory control mechanisms

• Studying how CA4 distribution changes in respiratory conditions like chronic obstructive pulmonary disease or pulmonary hypertension

• Exploring the relationship between CA4 activity and ciliary motility in respiratory epithelium, as recent research has shown decreased ciliary motility in CO₂/HCO₃⁻-free solutions

Renal Applications:

• Characterizing CA4's contribution to proximal tubular acidification processes

• Mapping the relationship between CA4 expression and transporter proteins involved in acid-base balance

• Investigating changes in CA4 expression in models of renal tubular acidosis or other acid-base disorders

• Studying the interplay between CA4 and other carbonic anhydrase isoforms in maintaining renal pH homeostasis

These applications can be enhanced by combining CA4 immunodetection with functional assays of enzyme activity and physiological measurements of acid-base parameters.

What emerging roles for CA4 in neuroscience are being explored?

CA4's connection to neurological conditions suggests emerging roles in neuroscience that warrant investigation:

• Retinitis pigmentosa mechanisms: The connection between CA4 mutations and RP17 form of retinitis pigmentosa opens avenues for investigating how CA4 contributes to retinal health and photoreceptor function . CA4 antibodies can help characterize expression patterns in retinal tissues and track changes in models of retinal degeneration.

• Brain pH regulation: CA4 is expressed in brain tissue, particularly the cerebellum . Researchers can use CA4 antibodies to map its distribution across brain regions and investigate its role in regulating brain extracellular pH, which affects neuronal excitability and synaptic transmission.

• Cerebrovascular function: As a GPI-anchored enzyme on capillary luminal surfaces, CA4 may play important roles in cerebrovascular physiology . Immunohistochemical studies using CA4 antibodies can reveal its distribution across the blood-brain barrier and help elucidate its functions in cerebral blood flow regulation.

• Neuroinflammation: Given the expression of CA4 on activated immune cells in some contexts , researchers can explore whether neuroinflammatory conditions alter CA4 expression in brain-resident or infiltrating immune cells.

These emerging areas highlight CA4's potential significance beyond its classical roles in acid-base homeostasis, opening new research directions where CA4 antibodies serve as critical investigative tools.

How can CA4 antibody-based imaging be optimized for in vivo applications?

Advancing CA4 antibody applications toward in vivo imaging presents both challenges and opportunities:

• Antibody format optimization: Converting conventional CA4 antibodies to smaller formats like Fab fragments or single-chain variable fragments (scFvs) can improve tissue penetration and reduce immunogenicity for in vivo applications.

• Multimodal imaging approaches: Taking inspiration from bispecific antibody approaches used in cancer imaging , researchers can develop bispecific CA4 antibodies coupled with appropriate imaging moieties. This strategy has shown promise in other systems, such as the bsPAM4 antibody used for pancreatic cancer imaging .

• Targeted delivery systems: Conjugating CA4 antibodies to nanoparticles or liposomes containing contrast agents can enhance signal-to-noise ratios while maintaining target specificity.

• Developability assessment: When designing CA4 antibodies for in vivo applications, researchers should apply high-throughput developability workflows similar to those used for therapeutic antibodies . These approaches help identify candidates with optimal stability, low aggregation potential, and favorable pharmacokinetics.

• Species cross-reactivity considerations: Since preclinical studies often involve animal models, developing CA4 antibodies with cross-reactivity to both human and model organism CA4 would facilitate translational research.

These approaches can extend CA4 antibody applications beyond traditional ex vivo and in vitro research into more clinically relevant in vivo imaging contexts.