CCB4 Antibody

What is the optimal dilution range for CA4 antibody in Western blot applications?

Western blot experiments with CA4 antibody typically require careful optimization of antibody concentration. Based on validated protocols, 1 μg/mL of CA4 antibody followed by an appropriate HRP-conjugated secondary antibody (such as Anti-Goat IgG) provides specific detection of CA4 at approximately 33 kDa under reducing conditions . When performing initial optimization, create a dilution series (0.5-2 μg/mL) to determine the optimal concentration for your specific sample type. Consider that sample preparation significantly impacts results - using appropriate lysis buffers that efficiently extract membrane-bound proteins is essential for CA4 detection. Always include positive control tissues such as human lung or brain (cerebellum) lysates to validate antibody performance in each experiment.

What are the most reliable positive control tissues for validating CA4 antibody specificity?

Selecting appropriate positive controls is critical for validating CA4 antibody performance. Human tissues with documented CA4 expression include:

• Human lung tissue - Shows consistent CA4 expression in pulmonary capillaries

• Human brain (cerebellum) tissue - Demonstrates reliable CA4 detection at approximately 33 kDa by Western blot

• Human kidney tissue - Exhibits CA4 expression, particularly in proximal tubules

• Human colon tissue - Shows specific membrane localization in epithelial cells of mucosal glands

Additionally, the A549 human lung carcinoma cell line has been validated for CA4 expression and can serve as a cellular positive control . When validating a new CA4 antibody lot, comparing staining patterns and band intensity across these validated positive controls ensures reliability and consistency between experiments.

How should sample preparation be modified for optimal CA4 antibody performance in different applications?

CA4's nature as a GPI-anchored membrane protein necessitates specific sample preparation considerations across different experimental applications:

For Western blotting:

• Use lysis buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40) to efficiently solubilize membrane proteins

• Process samples under reducing conditions as specified in validated protocols

• Avoid excessive heating which may cause aggregation of membrane proteins

For immunohistochemistry:

• Implement heat-induced epitope retrieval using basic pH retrieval solution for paraffin-embedded sections

• Allow sufficient permeabilization time to ensure antibody access to membrane-bound epitopes

• Consider using fresh-frozen sections for applications where epitope sensitivity to fixation is a concern

For immunoprecipitation:

• Select lysis buffers with gentler detergents (0.5-1% digitonin or 0.5% CHAPS) to preserve protein-protein interactions

• Include protease inhibitors to prevent degradation during extraction

• Pre-clear lysates thoroughly to reduce non-specific binding

These modifications account for CA4's membrane localization and ensure optimal detection across different experimental platforms.

How can CA4 antibody be utilized to investigate the relationship between CA4 and ciliary motility in epithelial cells?

Investigating CA4's role in ciliary motility requires integrated application of CA4 antibodies across multiple techniques. Studies have demonstrated that CO2/HCO3--free solutions decrease ciliary motility in human nasal epithelial cells with elevated pH, a process involving CA4 . To examine this relationship:

• Immunocytochemistry: Utilize CA4 antibody to determine subcellular localization in ciliated epithelial cells, establishing spatial relationships between CA4 and ciliary structures. Use confocal microscopy for co-localization studies with ciliary markers.

• Functional correlation analysis: Combine CA4 antibody detection with real-time measurements of ciliary beat frequency under controlled pH and bicarbonate conditions. This approach helps establish direct correlations between CA4 expression levels and functional ciliary parameters.

• Mechanistic investigation: Employ CA4 antibody in combination with phospho-specific antibodies to examine how CA4-mediated pH regulation influences signaling pathways that control ciliary dynamics, particularly focusing on calcium-dependent mechanisms.

• Comparative analysis across conditions: Apply CA4 antibody to quantify expression differences between normal and pathological specimens (e.g., chronic rhinosinusitis), correlating expression levels with clinical or functional parameters.

This multi-faceted approach provides comprehensive insights into CA4's regulatory role in ciliary function across different physiological and pathological contexts.

What are the methodological differences when using CA4 antibody in Western blot versus Simple Western™ assays?

CA4 antibody application in traditional Western blot versus automated Simple Western™ assays reveals important methodological differences researchers should consider:

The molecular weight difference (33 kDa vs. 40 kDa) likely reflects differences in sample preparation affecting post-translational modifications or protein migration characteristics . The substantially higher antibody concentration required for Simple Western™ (10 μg/mL vs. 1 μg/mL) suggests different detection sensitivity thresholds between methods. These differences highlight the importance of method-specific optimization when transitioning between platforms and the need to establish specific positive controls for each system.

How can immunohistochemistry with CA4 antibody be optimized for detecting membrane-bound CA4 in paraffin-embedded tissues?

Optimizing immunohistochemistry for membrane-bound CA4 in paraffin-embedded tissues requires attention to several critical parameters:

• Epitope retrieval: Heat-induced epitope retrieval using basic pH buffer is essential. Validated protocols use specialized reagents like VisUCyte Antigen Retrieval Reagent-Basic to effectively expose the CA4 epitope while maintaining tissue morphology .

• Antibody concentration and incubation: For optimal membrane staining, a concentration of 3 μg/mL with 1-hour room temperature incubation has been validated. This provides sufficient sensitivity while minimizing background .

• Detection system selection: Polymer-based detection systems (such as Anti-Goat IgG VisUCyte™ HRP Polymer Antibody) provide superior signal amplification for membrane proteins compared to standard ABC methods, enhancing sensitivity for the relatively low-abundance CA4 .

• Signal interpretation: When properly optimized, CA4 staining should localize specifically to cell membranes, particularly in epithelial cells of structures like colon mucosal glands. Diffuse cytoplasmic staining may indicate poor fixation or non-specific binding .

• Counterstaining: Light hematoxylin counterstaining provides optimal visualization of membrane-localized CA4 without obscuring the specific DAB signal.

Following these optimization steps ensures reliable detection of membrane-bound CA4 while minimizing background and non-specific staining artifacts.

How should I troubleshoot inconsistent CA4 antibody staining patterns in immunohistochemistry?

Inconsistent CA4 antibody staining patterns in immunohistochemistry can stem from multiple sources. Implement this systematic troubleshooting approach:

• Fixation assessment:

  • Overfixation can mask the CA4 epitope - standardize fixation time (24-48 hours in 10% neutral buffered formalin)

  • Underfixation can cause tissue degradation - ensure adequate fixation penetration

  • Consider testing different fixatives if problems persist

• Antigen retrieval optimization:

  • CA4 epitope requires heat-induced retrieval with basic pH buffer

  • Test multiple retrieval durations (10-30 minutes)

  • Compare different retrieval methods (microwave, pressure cooker, water bath)

  • Ensure complete coverage of sections with retrieval solution

• Antibody validation:

  • Test current antibody lot on validated positive control tissue (human colon shows reliable membrane staining)

  • Compare with previous lots if available

  • Consider testing alternative CA4 antibodies targeting different epitopes

• Background reduction:

  • Implement additional blocking steps (avidin/biotin, protein blocking)

  • Add 0.1-0.3% Triton X-100 to wash buffers

  • Include 1-3% normal serum from the secondary antibody species in antibody diluent

  • Increase washing duration and frequency between steps

• Detection system considerations:

  • Ensure secondary antibody compatibility with host species of CA4 antibody

  • Optimize enzyme substrate development time

  • Consider polymer-based detection systems for enhanced sensitivity

Systematically addressing each parameter while maintaining consistent positive controls between experiments will help identify and resolve the source of staining inconsistencies.

What are the key considerations when designing experiments to investigate CA4's potential role in disease pathogenesis?

Designing robust experiments to investigate CA4's role in disease pathogenesis requires careful experimental planning:

• Case-control tissue selection:

  • Match cases and controls for age, sex, and relevant clinical parameters

  • Consider tissue microarrays for high-throughput screening across multiple specimens

  • Include gradient of disease severity to assess correlation with CA4 expression

• Multimodal CA4 detection:

  • Quantify protein levels using validated Western blot protocols (1 μg/mL antibody concentration)

  • Assess spatial distribution through immunohistochemistry (3 μg/mL concentration)

  • Measure mRNA expression via qRT-PCR or RNA-seq to distinguish transcriptional vs. post-transcriptional regulation

• Functional correlation:

  • Link CA4 expression to enzymatic activity using carbonic anhydrase activity assays

  • Correlate CA4 levels with physiological parameters (e.g., tissue pH, bicarbonate levels)

  • Assess downstream signaling pathway activation

• Genetic association analysis:

  • Screen for CA4 mutations in patient populations (particularly examining the Arg14Trp mutation linked to retinitis pigmentosa)

  • Consider genotype-phenotype correlation studies

  • Implement CRISPR-Cas9 editing to model specific mutations in cell systems

• Intervention studies:

  • Design CA4 knockdown/overexpression experiments in relevant cell models

  • Utilize carbonic anhydrase inhibitors with varying isoform selectivity

  • Assess phenotypic rescue with wild-type CA4 in mutant backgrounds

• Controls and validation:

  • Include tissue-specific positive controls for CA4 expression (lung, brain, kidney)

  • Validate antibody specificity in each experimental system

  • Implement appropriate statistical analysis for sample size determination

This comprehensive experimental design allows for thorough investigation of CA4's potential mechanistic contributions to disease pathogenesis across multiple levels of analysis.

How can CA4 antibody be incorporated into multiplexed immunofluorescence panels for simultaneous detection of multiple markers?

Incorporating CA4 antibody into multiplexed immunofluorescence panels requires strategic planning to maintain signal specificity and quality:

• Panel design considerations:

  • The goat-derived CA4 antibody pairs well with rabbit, mouse, or rat antibodies in multiplex panels

  • Assign CA4 antibody a fluorophore with brightness appropriate for its expression level

  • Consider the subcellular localization (membrane) when selecting other markers to minimize spatial overlap

• Technical optimization:

  • Perform antibody titration in single-color controls before multiplexing

  • Starting with 3 μg/mL concentration (validated for IHC), adjust based on fluorescence signal strength

  • Test multiple epitope retrieval conditions to find optimal compatibility with other antibodies

  • Implement sequential staining with intermediate fixation if cross-reactivity occurs

• Signal balancing strategies:

  • Adjust individual antibody concentrations to achieve comparable signal intensities

  • Consider signal amplification systems (tyramide, dendrimer-based) for weaker signals

  • Optimize exposure settings during image acquisition for each channel

• Validation requirements:

  • Single-color controls to confirm specificity and expected localization pattern

  • Fluorescence-minus-one (FMO) controls to evaluate spectral overlap

  • Comparison of multiplex staining pattern with single-plex results

• Image acquisition and analysis:

  • Use sequential scanning to minimize bleed-through

  • Implement appropriate unmixing algorithms for closely overlapping fluorophores

  • Establish quantification parameters that account for membrane-localized signal

• Quality control measures:

  • Include consistent positive control tissue sections (human colon shows reliable membrane staining)

  • Maintain consistent threshold settings during analysis

  • Document lot numbers and antibody performance characteristics

This approach enables robust incorporation of CA4 antibody into multiplexed panels while maintaining specificity and quantitative reliability across experiments.

How can CA4 antibody be utilized to investigate the connection between CA4 mutations and retinitis pigmentosa?

The genetic mutation of CA4 (Arg14Trp in the signal peptide) cosegregates with the RP17 form of retinitis pigmentosa . Investigating this connection with CA4 antibody requires specialized approaches:

• Comparative expression analysis:

  • Apply CA4 antibody in Western blot (1 μg/mL) and immunohistochemistry (3 μg/mL) to compare expression patterns between normal and RP17-affected retinal tissues

  • Quantify expression differences in specific retinal layers and cell types

  • Correlate expression patterns with histopathological changes in the retina

• Cellular trafficking studies:

  • Develop in vitro models expressing wild-type versus R14W mutant CA4

  • Use CA4 antibody in immunofluorescence to trace subcellular localization

  • Employ super-resolution microscopy to detect subtle differences in membrane localization

  • Combine with ER/Golgi markers to identify potential trafficking defects

• Functional assessment:

  • Apply CA4 antibody in co-immunoprecipitation experiments to identify altered protein interactions in mutant CA4

  • Compare enzymatic activity of immunoprecipitated wild-type versus mutant CA4

  • Correlate CA4 localization with local pH regulation in retinal cells

• Animal model validation:

  • Develop transgenic models expressing the R14W mutation

  • Apply CA4 antibody to trace expression patterns during retinal development

  • Correlate CA4 expression with functional electroretinography (ERG) measurements

  • Evaluate potential therapeutic interventions through restoration of proper CA4 expression/function

This multifaceted approach can establish mechanistic links between the CA4 mutation and retinal degeneration, potentially identifying novel therapeutic targets for this genetic disease.

What experimental approaches can determine CA4's role in regulating pH homeostasis in respiratory epithelium?

CA4's function in regulating pH homeostasis in respiratory epithelium requires sophisticated experimental approaches using CA4 antibody:

• Expression mapping in respiratory epithelium:

  • Apply CA4 antibody (3 μg/mL) in immunohistochemistry to map expression patterns across different regions of respiratory tract

  • Use double immunofluorescence to co-localize CA4 with epithelial cell type markers

  • Compare expression patterns between healthy and diseased respiratory tissues

• Functional pH imaging studies:

  • Correlate CA4 antibody staining with pH-sensitive fluorescent probe measurements

  • Develop live cell imaging techniques combining CA4 immunolabeling with real-time pH monitoring

  • Implement microelectrode measurements of surface pH in conjunction with CA4 mapping

• Genetic manipulation approaches:

  • Design siRNA or CRISPR-based CA4 knockdown in primary respiratory epithelial cultures

  • Validate knockdown efficiency using CA4 antibody in Western blot (1 μg/mL)

  • Measure pH changes and bicarbonate transport in knockdown versus control cells

• Mechanistic pathway analysis:

  • Use CA4 antibody to immunoprecipitate protein complexes involved in bicarbonate transport

  • Identify CA4 interaction partners in respiratory epithelium

  • Correlate CA4 expression with ciliary beat frequency under varying pH conditions

• Disease model applications:

  • Compare CA4 expression and localization in models of respiratory diseases (asthma, COPD)

  • Correlate expression changes with functional outcomes

  • Assess potential therapeutic approaches targeting CA4 activity

This experimental framework provides comprehensive insights into CA4's physiological role in respiratory epithelial pH regulation and its potential contributions to respiratory pathologies.

How can CA4 antibody be integrated into studies of tumor microenvironment pH regulation?

Integrating CA4 antibody into tumor microenvironment pH regulation studies requires specialized experimental design:

• Expression profiling across tumor types:

  • Apply CA4 antibody in tissue microarrays spanning multiple tumor types

  • Compare CA4 expression between tumor centers and invasive margins

  • Correlate expression with hypoxia markers and patient outcomes

• Spatial pH mapping in relation to CA4 expression:

  • Combine CA4 immunohistochemistry with pH-sensitive probe imaging in serial sections

  • Develop multiplex immunofluorescence panels including CA4 (starting at 3 μg/mL) with hypoxia markers (HIF-1α, CAIX) and pH-regulatory proteins

  • Implement computational spatial analysis to correlate CA4 expression with regional pH variations

• Functional manipulation in tumor models:

  • Generate CA4 knockdown/overexpression in tumor cell lines

  • Validate manipulation using CA4 antibody in Western blot (1 μg/mL)

  • Measure extracellular acidification rates and response to metabolic challenges

  • Assess impact on tumor growth and metastatic potential in vivo

• Therapeutic targeting approaches:

  • Screen carbonic anhydrase inhibitors with varying selectivity for CA4

  • Monitor changes in CA4 expression and localization during treatment

  • Combine with immune checkpoint inhibitors to assess potentiation of immunotherapy

• Single-cell analysis:

  • Implement imaging mass cytometry incorporating CA4 antibody

  • Characterize CA4-expressing cells within heterogeneous tumor microenvironments

  • Correlate with metabolic markers and immune cell infiltration patterns

This integrated approach provides insights into CA4's role in tumor pH regulation and identifies potential therapeutic opportunities targeting tumor acidification mechanisms.

What novel methodologies are emerging for CA4 antibody applications in single-cell analysis?

Emerging single-cell methodologies incorporating CA4 antibody offer unprecedented insights into cellular heterogeneity:

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated CA4 antibody enables simultaneous measurement with 40+ markers

  • Correlation of CA4 expression with cell lineage markers and functional states

  • High-dimensional clustering algorithms reveal previously unidentified CA4+ cell subpopulations

• Single-cell spatial transcriptomics integration:

  • CA4 antibody immunofluorescence combined with in situ RNA sequencing

  • Correlation of protein expression with transcriptional programs

  • Spatial mapping of CA4+ cells within tissue microenvironments

• Microfluidic-based single-cell Western blotting:

  • Adaptation of validated CA4 antibody protocols (1 μg/mL) to microfluidic platforms

  • Quantification of CA4 protein levels in individual cells

  • Correlation with other pH-regulatory proteins at single-cell resolution

• Proximity ligation assays:

  • Combining CA4 antibody with antibodies against potential interaction partners

  • Detection of protein-protein interactions in situ

  • Spatial mapping of functional CA4 complexes at subcellular resolution

• Live-cell antibody fragment applications:

  • Development of non-perturbing Fab fragments or nanobodies derived from CA4 antibodies

  • Real-time tracking of CA4 dynamics in living cells

  • Correlation with pH fluctuations using genetically-encoded pH sensors

These emerging methodologies expand CA4 antibody applications beyond traditional approaches, offering unprecedented insights into CA4 biology at single-cell resolution with spatial context maintained.

How can computational image analysis enhance CA4 antibody-based research?

Advanced computational image analysis significantly enhances the value of CA4 antibody-based research:

• Automated membrane localization quantification:

  • Develop algorithms specifically designed to identify and quantify membrane-localized CA4 staining

  • Implement membrane segmentation tools to distinguish true membrane signal from cytoplasmic background

  • Create standardized metrics for membrane staining intensity and continuity

• Multi-parameter correlation analysis:

  • Combine CA4 antibody staining with multiple additional markers

  • Implement colocalization algorithms with statistical validation

  • Develop neighborhood analysis to identify cell types adjacent to CA4+ cells

• Machine learning classification approaches:

  • Train neural networks to identify specific CA4 staining patterns

  • Develop automated quality control for immunohistochemistry results

  • Implement unbiased classification of normal versus aberrant staining patterns

• 3D reconstruction techniques:

  • Apply CA4 antibody in serial section imaging or tissue clearing protocols

  • Develop 3D reconstruction algorithms to visualize CA4 distribution throughout entire tissue volumes

  • Quantify spatial relationships in three dimensions

• Large-scale image database integration:

  • Standardize CA4 antibody staining quantification across multiple research sites

  • Develop normalization algorithms to account for technical variation

  • Create accessible repositories of validated CA4 staining patterns

These computational approaches transform qualitative CA4 antibody staining into quantitative, reproducible data while revealing patterns and relationships not detectable through visual assessment alone.

What are the future prospects for combining CA4 antibody approaches with genetic manipulation technologies?

The integration of CA4 antibody approaches with advanced genetic manipulation technologies offers powerful new research paradigms:

• CRISPR-based genome editing validation:

  • Use CA4 antibody in Western blot (1 μg/mL) and immunofluorescence (3 μg/mL) to validate CRISPR-mediated CA4 knockout or mutation

  • Develop quantitative assessment of editing efficiency at protein level

  • Correlate protein expression changes with functional outcomes

• Conditional genetic systems:

  • Design tissue-specific or inducible CA4 knockout/knockin models

  • Validate spatiotemporal expression patterns using CA4 antibody

  • Track dynamic changes in CA4 expression during development or disease progression

• Base editing and prime editing applications:

  • Introduce precise CA4 mutations modeling human variants (like R14W associated with retinitis pigmentosa)

  • Validate editing outcomes at protein level using CA4 antibody

  • Assess functional consequences of specific amino acid changes

• Single-cell genetic perturbation analysis:

  • Combine CRISPR screens with CA4 antibody-based protein detection

  • Identify genetic modifiers of CA4 expression or localization

  • Implement pooled genetic screens with CA4 protein-level readouts

• In vivo genetic manipulation monitoring:

  • Track CA4 expression dynamics following in vivo genetic manipulation

  • Develop in vivo imaging approaches using labeled CA4 antibody derivatives

  • Correlate genetic manipulation with physiological outcomes

• Therapeutic gene editing assessment:

  • Validate gene therapy approaches for CA4-related diseases using antibody-based detection

  • Monitor off-target effects on CA4 expression from other genetic manipulations

  • Develop quality control metrics for therapeutic editing of CA4

This integration creates powerful research platforms that connect genetic manipulation directly to protein-level outcomes, accelerating mechanistic understanding and therapeutic development for CA4-related conditions.

How do CA4 antibody applications compare with antibodies targeting other carbonic anhydrase isoforms?

Antibodies targeting different carbonic anhydrase isoforms have distinct applications reflecting their unique biology:

These differences necessitate specific methodological adaptations when transitioning between CA isoform studies. The membrane localization of CA4 requires particular attention to extraction methods and immunohistochemical protocols compared to cytosolic isoforms. Understanding these distinctions enables researchers to select appropriate antibodies and methods aligned with their specific research questions about carbonic anhydrase biology.

What are the most significant recent methodological advances in CA4 antibody applications?

Recent methodological advances have significantly expanded CA4 antibody research capabilities:

• Automated high-throughput analysis systems:

  • Simple Western™ automated capillary immunoassay systems enable standardized CA4 detection at approximately 40 kDa with 10 μg/mL antibody concentration

  • Increased throughput with reduced sample volume requirements

  • Enhanced reproducibility through automation of separation and detection steps

• Enhanced visualization systems:

  • Advanced polymer detection chemistries like VisUCyte™ HRP Polymer improve signal-to-noise ratio for membrane-bound CA4

  • Super-resolution microscopy techniques reveal CA4 distribution within membrane microdomains

  • Tissue clearing protocols enable whole-organ 3D visualization of CA4 distribution

• Multiplex detection platforms:

  • Integration of CA4 antibody into multiplexed immunofluorescence panels

  • Development of spectral imaging systems reducing fluorophore cross-talk

  • Mass cytometry adaptation for single-cell protein profiling

• Quantitative image analysis:

  • Automated membrane quantification algorithms specific for GPI-anchored proteins

  • Machine learning approaches for pattern recognition in CA4 distribution

  • Large-scale image analysis across tissue microarrays

• Standardization initiatives:

  • Development of recombinant CA4 protein standards for absolute quantification

  • Implementation of digital pathology guidelines for reproducible CA4 assessment

  • Establishment of validated positive controls for different applications

These methodological advances collectively enhance sensitivity, specificity, throughput, and reproducibility of CA4 antibody applications, enabling more sophisticated investigations into CA4 biology in health and disease.

What are the key considerations when interpreting studies using CA4 antibodies in the scientific literature?

Critical evaluation of scientific literature using CA4 antibodies requires attention to several key methodological considerations:

• Antibody validation assessment:

  • Evaluate whether proper positive controls were used (human brain cerebellum, lung, kidney, or colon tissues)

  • Check if expected molecular weight was demonstrated (approximately 33 kDa in Western blot, 40 kDa in Simple Western)

  • Assess whether membrane localization was confirmed in immunohistochemistry applications

• Technical protocol evaluation:

  • Verify whether appropriate antibody concentrations were used (1 μg/mL for Western blot, 3 μg/mL for IHC, 10 μg/mL for Simple Western)

  • Confirm if proper epitope retrieval methods were employed (heat-induced with basic pH for paraffin sections)

  • Assess if membrane protein extraction methods were sufficient for CA4's GPI-anchored nature

• Specificity controls:

  • Look for inclusion of negative controls (isotype antibodies, CA4-negative tissues)

  • Check whether antibody specificity was validated through genetic approaches (siRNA, CRISPR knockout)

  • Evaluate if potential cross-reactivity with other CA isoforms was addressed

• Quantification methodology:

  • Assess whether appropriate quantification methods were used for membrane proteins

  • Verify statistical approaches for comparing CA4 expression across experimental groups

  • Check for transparent reporting of image acquisition and analysis parameters

• Biological context consideration:

  • Evaluate whether CA4's known biology as a GPI-anchored membrane enzyme was appropriately considered

  • Assess if experimental conditions might alter CA4 expression or localization

  • Consider whether findings align with known functions of CA4 in CO2 sensing and pH regulation