BCA4 Antibody

What is Carbonic Anhydrase IV (CA4) and why is it significant in research?

Carbonic Anhydrase IV (CA4) is a membrane-bound isozyme of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide to bicarbonate. It plays critical roles in pH regulation, ion transport, and gas exchange across membranes. CA4 is particularly important in research due to its involvement in respiratory and renal physiology, and potential implications in various pathological conditions. The protein is predominantly expressed on the luminal surfaces of certain endothelial cells, particularly in lung tissue where it facilitates carbon dioxide exchange . Understanding CA4's function requires specific antibodies that can distinguish it from other carbonic anhydrase isozymes, making CA4 antibodies essential tools in physiological and pathological investigations.

How can I verify the specificity of CA4 antibody in my experimental system?

Verifying CA4 antibody specificity requires a multi-step approach:

• Cross-reactivity analysis: Human CA4 antibodies may show approximately 10% cross-reactivity with recombinant human CA1, CA2, and recombinant mouse CA4, but typically do not cross-react with CA3, 8, 9, 10, 12, 13, or 14 . Always validate these patterns in your experimental system.

• Western blot validation: Perform Western blots with positive control tissues known to express CA4 (e.g., human lung tissue) and negative controls. For human samples, a specific band for CA4 should be detected at approximately 35 kDa under non-reducing conditions .

• Knockout/knockdown controls: Where possible, include CA4-knockout or knockdown samples to confirm antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with purified CA4 protein before application to your samples. Signal disappearance confirms specificity.

• Multiple antibody comparison: Use antibodies raised against different epitopes of CA4 to confirm consistent staining patterns.

What are the optimal sample preparation methods for CA4 antibody applications?

Sample preparation methods vary by application and tissue type:
For Western Blot analysis:

• Use non-reducing conditions when possible, as the CA4 antibody detection may be affected by reducing agents .

• Prepare lysates from fresh tissues or cultured cells using buffer systems compatible with membrane proteins (e.g., RIPA buffer with protease inhibitors).

• For membrane-bound CA4, ensure adequate membrane solubilization with appropriate detergents.
  For Immunohistochemistry:

• Both paraffin-embedded and frozen sections can be suitable, depending on the specific antibody .

• For paraffin sections, optimize antigen retrieval methods (heat-induced or enzymatic) to expose the CA4 epitope.

• Consider mild fixation protocols to preserve membrane protein epitopes.
  For Flow Cytometry:

• Use gentle cell dissociation methods to preserve membrane integrity.

• Optimize permeabilization protocols if targeting intracellular domains.

How can I optimize CA4 antibody for dual labeling experiments with other carbonic anhydrase isozymes?

Dual labeling experiments with CA4 and other carbonic anhydrase isozymes require careful planning:

• Antibody species selection: Choose primary antibodies raised in different host species (e.g., mouse anti-CA4 and rabbit anti-CA2) to allow simultaneous detection with species-specific secondary antibodies.

• Cross-reactivity mitigation: Given the 10% cross-reactivity of some CA4 antibodies with CA1 and CA2 , implement additional controls:

  • Include single-stained samples for each antibody

  • Perform sequential staining with complete washing between steps

  • Consider using directly conjugated primary antibodies

• Signal separation: If using fluorescence detection:

  • Select fluorophores with minimal spectral overlap

  • Include appropriate compensation controls

  • Consider linear unmixing algorithms for closely overlapping signals

• Validation strategy: Confirm co-localization or distinct localization patterns using:

  • High-resolution confocal microscopy

  • Super-resolution techniques for membrane-bound isozymes

  • Correlative electron microscopy for subcellular localization

What experimental approaches can resolve contradictory data when studying CA4 interactions with monocarboxylate transporters?

When faced with contradictory data regarding CA4 interactions with monocarboxylate transporters:

• Protein complex isolation techniques:

  • Implement proximity ligation assays (PLA) to verify direct protein interactions

  • Use chemical crosslinking followed by immunoprecipitation to stabilize transient interactions

  • Apply FRET/BRET approaches to investigate real-time interactions in live cells

• Functional validation:

  • Measure transport activity with and without CA4 inhibition

  • Perform site-directed mutagenesis of key CA4 residues to identify interaction domains

  • Use domain-swapping experiments between CA isozymes to define specificity determinants

• Context-dependent analysis:

  • Systematically test interactions under varying pH conditions

  • Assess the influence of membrane microdomains on interaction strength

  • Evaluate the role of accessory proteins like CD147 and GP70 in mediating interactions

• Model systems comparison:

  • Compare results between different cell lines, primary cultures, and tissue preparations

  • Validate findings in CA4-knockout models and rescue experiments

  • Consider species-specific differences in CA4 structure and function

How can computational modeling inform CA4 antibody epitope design for improved specificity?

Computational modeling can significantly enhance CA4 antibody epitope design:

• Structural bioinformatics approaches:

  • Perform sequence alignment of CA isozymes to identify CA4-unique regions

  • Use 3D structural models to identify surface-exposed, CA4-specific epitopes

  • Implement molecular dynamics simulations to assess epitope accessibility

• Biophysics-informed modeling:

  • Apply machine learning algorithms trained on experimentally selected antibodies to predict epitope-paratope interactions

  • Identify distinct binding modes associated with specific ligands

  • Generate antibody variants with customized specificity profiles not present in initial libraries

• Experimental validation pipeline:

  • Design phage display experiments with systematic variation of complementary determining regions (CDRs)

  • Validate computationally predicted epitopes using alanine scanning mutagenesis

  • Implement iterative design-build-test cycles to refine antibody specificity

What are the optimal protocols for detecting CA4 expression in different tissue types?

Detection protocols for CA4 expression must be tailored to specific tissue types:
For Lung Tissue:

• Use 2 μg/mL of Mouse Anti-Human CA4 Monoclonal Antibody for optimal Western blot detection

• Implement non-reducing conditions for sample preparation

• Apply HRP-conjugated Anti-Mouse IgG Secondary Antibody for visualization
  For Renal Tissue:

• Consider membrane fractionation to enrich for CA4

• Implement mild fixation protocols for immunohistochemistry to preserve membrane epitopes

• Use lower antibody concentrations (0.5-1 μg/mL) to minimize background
  For Endothelial Cells:

• Optimize cell lysis conditions with specialized membrane protein extraction buffers

• Consider confocal microscopy to confirm membrane localization

• Implement flow cytometry with non-permeabilizing conditions to detect surface expression

How should researchers interpret variations in CA4 molecular weight across different experimental systems?

Variations in CA4 molecular weight across experimental systems require systematic analysis:

• Expected molecular weight patterns:

  • Human CA4 typically appears at approximately 35 kDa under non-reducing conditions

  • Glycosylation modifications may result in bands between 35-40 kDa

  • Proteolytic processing may generate lower molecular weight forms

• Resolution strategies for unexpected patterns:

  • Compare reducing vs. non-reducing conditions to assess disulfide bond contributions

  • Perform deglycosylation experiments with PNGaseF to identify glycosylation contributions

  • Use N-terminal and C-terminal targeted antibodies to identify truncated forms

• Sample preparation considerations:

  • Assess the impact of different detergents on solubilization efficiency

  • Evaluate the effect of protease inhibitor cocktails on band patterns

  • Consider the influence of freeze-thaw cycles on protein integrity

What approaches can be used to quantify CA4 antibody binding kinetics for research applications?

Quantifying CA4 antibody binding kinetics requires specialized techniques:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified CA4 protein on a sensor chip

  • Measure real-time binding of antibody at different concentrations

  • Calculate association (kon) and dissociation (koff) rate constants

  • Determine equilibrium dissociation constant (KD) as koff/kon

• Bio-Layer Interferometry (BLI):

  • Attach antibody to biosensor tips

  • Measure association and dissociation phases with purified CA4

  • Compare binding kinetics with other CA isozymes to confirm specificity

  • Assess the impact of buffer conditions on binding parameters

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG)

  • Evaluate the contribution of different binding forces to complex stability

How can CA4 antibodies be effectively incorporated into antibody-drug conjugates (ADCs) for targeted therapy research?

Incorporating CA4 antibodies into ADCs requires specialized approaches:

• Conjugation strategy selection:

  • Evaluate random lysine-linked conjugation vs. site-specific methods

  • Consider glycosite-specific conjugation approaches for consistent Drug-Antibody Ratio (DAR)

  • Assess the impact of conjugation on CA4 binding activity

• DAR optimization and monitoring:

  • Implement rapid DAR analysis methods using robust deglycosylation and LC-MS detection

  • Optimize conjugation conditions to achieve targeted DAR values

  • Monitor DAR stability during storage and under physiological conditions

• Functional validation:

  • Assess binding specificity before and after conjugation

  • Evaluate internalization kinetics in CA4-expressing cells

  • Measure drug release in lysosomal conditions

• Therapeutic potential evaluation:

  • Test efficacy in models where CA4 is overexpressed

  • Assess potential off-target effects based on known cross-reactivity patterns

  • Compare with other targeting strategies for the same indication

What are the critical considerations when applying CA4 antibodies in single-cell analysis techniques?

Single-cell analysis with CA4 antibodies requires specific technical considerations:

• Single-cell protein detection methods:

  • Optimize antibody concentrations for mass cytometry (CyTOF) applications

  • Develop custom antibody conjugates with appropriate metal isotopes or fluorophores

  • Validate staining protocols using known CA4-positive and negative cell populations

• Spatial transcriptomics integration:

  • Combine CA4 antibody staining with mRNA detection

  • Correlate protein expression with transcript levels at single-cell resolution

  • Identify potential post-transcriptional regulation mechanisms

• Functional correlation:

  • Integrate CA4 detection with physiological readouts (e.g., pH indicators)

  • Link expression levels with cell-specific functions

  • Assess heterogeneity of CA4 expression within seemingly homogeneous cell populations

How can researchers troubleshoot unexpected subcellular localization patterns when using CA4 antibodies?

When troubleshooting unexpected CA4 localization patterns:

• Antibody validation approaches:

  • Confirm epitope specificity using peptide competition assays

  • Test multiple antibodies targeting different CA4 domains

  • Validate with genetically tagged CA4 (e.g., GFP fusion) as reference

• Sample preparation optimization:

  • Evaluate different fixation and permeabilization methods

  • Compare detergent types and concentrations for membrane protein preservation

  • Assess the impact of antigen retrieval techniques on epitope accessibility

• Advanced imaging techniques:

  • Apply super-resolution microscopy for detailed subcellular localization

  • Use correlative light and electron microscopy (CLEM) for ultrastructural context

  • Implement live-cell imaging with minimally disruptive labeling methods

• Biological interpretation strategies:

  • Consider developmental or physiological state-dependent localization changes

  • Evaluate the impact of cell activation or stress on CA4 trafficking

  • Assess potential post-translational modifications affecting localization

What statistical approaches are most appropriate for analyzing variable CA4 expression across tissue samples?

When analyzing variable CA4 expression across tissue samples:

How can researchers integrate CA4 antibody data with multi-omics approaches for comprehensive functional analysis?

Integrating CA4 antibody data with multi-omics approaches requires:

• Data integration strategies:

  • Correlate CA4 protein levels with transcriptomic profiles

  • Integrate with proteomic data to identify interaction networks

  • Combine with metabolomic data to assess functional impact on carbonic acid metabolism

• Computational analysis approaches:

  • Apply pathway enrichment analysis incorporating CA4-related functions

  • Implement network analysis to identify functional modules

  • Use machine learning algorithms to identify predictive biomarker signatures

• Validation experimental design:

  • Design targeted validation experiments for key computational predictions

  • Implement CRISPR-based functional genomics to validate pathway connections

  • Use small molecule inhibitors of CA4 to confirm functional relationships

What are best practices for reporting CA4 antibody validation in scientific publications?

Best practices for reporting CA4 antibody validation include:

• Essential reporting elements:

  • Provide complete antibody information: manufacturer, catalog number, clone ID, lot number

  • Detail the validation experiments performed specifically for your application

  • Include representative images of positive and negative controls

  • Report complete experimental conditions (concentrations, incubation times, buffers)

• Application-specific validation:

  • For Western blots: show full blots with molecular weight markers

  • For IHC/ICC: include appropriate positive and negative control tissues

  • For flow cytometry: provide gating strategies and fluorescence-minus-one controls

• Reproducibility considerations:

  • Describe number of experimental replicates and consistency between batches

  • Report all optimization steps performed

  • Consider depositing detailed protocols in repositories like protocols.io

• Resource sharing:

  • Provide information on reagent availability

  • Consider depositing validation data in antibody validation repositories

  • Reference the Research Resource Identifiers (RRIDs) for antibodies