CAR2 Antibody

What is CAR2 and what cellular functions does it regulate?

Carbonic Anhydrase II (CAR2) is an enzyme that catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons, playing a crucial role in pH regulation and fluid balance. In humans, the canonical CAR2 protein spans from Ser2 to Lys260 (Accession # P00918) and is approximately 27-30 kDa . CAR2 is the primary isoenzyme responsible for aqueous humor production in the eye and plays a major role in the regulation of intraocular pressure (IOP) . Within cellular contexts, CAR2 functions in various tissues with particularly high expression in heart, kidney, and epithelial tissues . The gene has been extensively studied in the context of glaucoma research due to its critical role in fluid dynamics of the eye .

What are the most reliable applications for CAR2 antibodies in research?

CAR2 antibodies have demonstrated efficacy in multiple experimental applications, with Western blot being the most widely used technique . Additional reliable applications include:

• Direct ELISA: Effective for quantitative detection of CAR2 in biological samples

• Immunohistochemistry (IHC): Allows visualization of CAR2 distribution in tissue sections

• Immunofluorescence: Enables subcellular localization studies and co-expression analyses

• Simple Western™: A capillary-based immunoassay system that can detect CAR2 at approximately 37 kDa under reducing conditions
  When selecting applications, researchers should consider that some CAR2 antibodies show approximately 5% cross-reactivity with recombinant human proteins in direct ELISAs, necessitating appropriate controls .

How should researchers differentiate between CAR2 and CARS2 antibodies?

Despite their similar nomenclature, CAR2 and CARS2 target entirely different proteins with distinct cellular functions:

What are the typical reactivity profiles for commercially available CAR2 antibodies?

Commercial CAR2 antibodies display varying species reactivity profiles:

• Human-specific: Most common and extensively validated

• Cross-reactive with rodent species: Many antibodies recognize mouse and rat CAR2

• Multi-species: Some antibodies demonstrate broader reactivity across species including human, mouse, rat, bovine, dog, guinea pig, and horse
  When selecting a CAR2 antibody, researchers should prioritize reagents with published validation in their species of interest. The sheep anti-human CAR2 antibody has been extensively validated for detection of human CAR2 in heart tissue, kidney tissue, and various human cell lines including Caki-2, A431, and Caco-2 .

What is the significance of CAR2 in eye physiology and glaucoma research?

CAR2 plays a critical role in ocular physiology as the primary enzyme responsible for aqueous humor production in the ciliary body. Recent research has established that:

• CAR2 gene expression is directly linked to the regulation of intraocular pressure (IOP)

• CAR2 knockout via CRISPR-Cas9 can significantly reduce IOP in both normal mice and glaucoma models by inhibiting aqueous humor production

• CAR2 inhibition has demonstrated potential to delay or halt glaucomatous damage induced by prolonged high IOP

• Gene-editing approaches targeting CAR2 may surpass the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide
  This emerging research highlights the importance of CAR2 antibodies for validating genetic manipulation and evaluating expression changes in experimental models of eye disease.

How can researchers optimize CAR2 antibody dilutions for Western blot applications?

Optimizing CAR2 antibody dilutions requires a systematic approach based on tissue-specific expression levels and antibody characteristics:

• Begin with manufacturer's recommended dilution ranges (typically 1-10 μg/mL for affinity-purified antibodies)

• Perform a dilution series using positive control samples with known CAR2 expression (kidney, heart tissues, or Caki-2, A431 cell lysates)

• Include appropriate negative controls and blocking peptides to distinguish specific from non-specific binding

• For Western blot applications, use reducing conditions and Immunoblot Buffer Group 1

• Expect CAR2 detection at approximately 27-30 kDa, though this may vary to 37 kDa in some systems

• When using Simple Western™ systems, load samples at approximately 0.2 mg/mL concentration
  The optimal antibody concentration should produce clear specific bands with minimal background. Validation across multiple tissue sources is recommended for confirming specificity.

What methodological considerations are important when using CAR2 antibodies to validate CRISPR-Cas9 gene editing?

When using CAR2 antibodies to validate CRISPR-Cas9 gene editing, researchers should implement a comprehensive validation strategy:

• Design a dual-target CRISPR system for efficient disruption of the CAR2 gene, as single-guide approaches may yield incomplete knockouts

• Use western blot with validated anti-CAR2 antibodies to quantify protein reduction rather than merely presence/absence

• Implement immunohistochemistry to confirm tissue-specific knockout, particularly in targeted areas like the ciliary body

• Compare CAR2 expression between edited and control samples using at least two independent antibodies targeting different epitopes

• Include time-course analysis, as protein persistence may extend beyond successful gene editing

• Correlate protein expression with functional outcomes (e.g., intraocular pressure measurements in ocular studies)
  This comprehensive approach provides stronger evidence of successful gene editing than relying solely on genetic sequencing of the target region.

How can CAR2 antibodies be effectively employed in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence with CAR2 antibodies requires careful planning to avoid technical pitfalls:

• Choose primary antibodies raised in different host species to prevent cross-reactivity

• When studying CAR2 in ocular tissues, combine with markers for:

  • Ciliary epithelium (AQP1 or sodium-potassium ATPase)

  • Trabecular meshwork (myocilin)

  • Retinal ganglion cells (RBPMS or Brn3a)

• Implement sequential staining protocols when using multiple antibodies from the same species

• Use spectral unmixing for channels with overlapping emission spectra

• Include single-stain controls to establish appropriate compensation settings

• Apply tissue-specific antigen retrieval methods optimized for preserving CAR2 epitopes

• Validate staining patterns through comparison with published literature
  This approach allows for contextual understanding of CAR2 expression relative to other markers critical in understanding ocular physiology and pathology.

What strategies can overcome common challenges in detecting CAR2 in formalin-fixed tissues?

Detection of CAR2 in formalin-fixed tissues can be challenging due to epitope masking. Researchers can implement these methodological improvements:

• Optimize antigen retrieval methods:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Protease-based retrieval for heavily fixed samples

• Extend primary antibody incubation to overnight at 4°C to improve penetration

• Utilize signal amplification systems such as tyramide signal amplification

• Test multiple CAR2 antibodies targeting different epitopes, as some may be more resistant to fixation effects

• Include positive control tissues with known high CAR2 expression (kidney, heart)

• Consider dual immunofluorescence with carbonic anhydrase inhibitor fluorescent probes to confirm specificity

• Implement automated staining platforms to ensure consistent antibody application and washing steps
  These approaches significantly improve detection sensitivity and specificity in archival tissue samples.

How can researchers interpret CAR2 expression differences between normal and pathological ocular tissues?

Interpreting CAR2 expression differences requires contextual analysis and consideration of several factors:

• Establish baseline CAR2 expression in multiple control samples to account for natural variation

• Quantify CAR2 levels using digital image analysis with appropriate normalization to housekeeping proteins

• In glaucoma models, correlate CAR2 expression with:

  • Intraocular pressure measurements

  • Structural changes in the trabecular meshwork

  • Retinal ganglion cell survival metrics

• Compare expression patterns with functional outcomes following CAR2 inhibition or genetic manipulation

• Account for potential compensatory upregulation of other carbonic anhydrase isoforms

• Consider post-translational modifications that may affect CAR2 function without altering total protein levels
  This comprehensive approach provides more valuable insights than simple presence/absence analysis, particularly when evaluating potential therapeutic targets.

What are the technical considerations when comparing results from different CAR2 antibody clones?

When comparing results from different CAR2 antibody clones, researchers should account for:

• Epitope differences:

  • N-terminal targeted antibodies may detect different isoforms than C-terminal targeted ones

  • Central domain antibodies may be more sensitive to conformational changes

• Clone-specific characteristics:

  • Monoclonal antibodies provide higher reproducibility but may be sensitive to epitope masking

  • Polyclonal antibodies offer broader epitope recognition but higher batch-to-batch variability

• Comparative validation approach:

  • Test multiple antibodies on identical sample sets

  • Document differences in sensitivity and background across tissue types

  • Verify molecular weight consistency across different detection systems

• Protocol standardization:

  • Maintain consistent blocking conditions, antibody dilutions, and incubation times

  • Use automated systems where possible to minimize technical variability

• Reporting standards:

  • Document complete antibody information including catalog number, lot number, and dilution

  • Report all optimization steps performed to enable reproducibility
    This methodical approach facilitates more accurate cross-study comparisons and improves research reproducibility.

How can researchers effectively use CAR2 antibodies to validate gene therapy approaches for glaucoma?

CAR2 antibodies serve as critical tools for validating gene therapy approaches targeting glaucoma through several methodological applications:

• Pre-treatment assessment:

  • Establish baseline CAR2 expression patterns in target tissues

  • Quantify protein levels for later comparative analysis

• Post-intervention validation:

  • Confirm successful ShH10 adenovirus-associated virus transduction and specificity for ciliary body targeting

  • Verify CAR2 protein reduction following CRISPR-Cas9 mediated gene knockout

• Functional correlation:

  • Associate CAR2 protein levels with measured changes in intraocular pressure

  • Compare knockout efficiency with the therapeutic effect of conventional carbonic anhydrase inhibitors

• Long-term monitoring:

  • Assess stability of reduced CAR2 expression over time following single-treatment intervention

  • Monitor for potential compensatory mechanisms through measurement of other carbonic anhydrase isoforms

• Experimental controls:

  • Include sham-treated controls and non-targeting CRISPR controls

  • Implement dual antibody validation using independent clones targeting different CAR2 epitopes
    This systematic approach provides comprehensive validation of gene therapy efficacy beyond simple genetic confirmation of target modification.

What controls are essential when using CAR2 antibodies for experimental validation?

A robust control strategy for CAR2 antibody experiments should include:

• Positive tissue controls:

  • Human kidney and heart tissues (high endogenous expression)

  • Caki-2, A431, and Caco-2 cell lines (validated CAR2 expression)

• Negative controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Tissues or cell lines with confirmed absence/knockdown of CAR2

  • Isotype controls matching the primary antibody species and class

• Validation controls:

  • Peptide competition assays using recombinant human CAR2 protein

  • siRNA or CRISPR knockdown samples to confirm antibody specificity

  • Cross-validation with alternative detection methods (mRNA quantification)

• Technical controls:

  • Loading controls for western blot (housekeeping proteins)

  • Tissue processing controls (fixation time, antigen retrieval)

  • Antibody titration series to determine optimal working concentration
    Implementation of these controls significantly enhances data reliability and reproducibility in CAR2 research.

How should researchers design experiments to distinguish between different carbonic anhydrase isoforms?

Distinguishing between carbonic anhydrase isoforms requires careful experimental design:

• Antibody selection criteria:

  • Choose antibodies raised against unique epitopes not conserved across isoforms

  • Verify specificity through testing against recombinant proteins of multiple isoforms

  • Consider using multiple antibodies targeting different regions of the same isoform

• Expression analysis approach:

  • Complement protein detection with isoform-specific mRNA quantification

  • Use subcellular fractionation to separate cytosolic (CAR2) from membrane-bound isoforms

  • Implement isoform-specific activity assays based on enzymatic properties

• Functional validation:

  • Utilize isoform-selective inhibitors in parallel experiments

  • Perform genetic knockdown of specific isoforms to confirm antibody specificity

  • Analyze tissue distribution patterns characteristic of particular isoforms

• Data interpretation:

  • Account for potential cross-reactivity percentages in quantitative analyses

  • Consider evolutionary conservation when working with non-human models

  • Assess molecular weight differences between isoforms (CAR2: 27-30 kDa vs. others)
    This multifaceted approach minimizes misinterpretation due to antibody cross-reactivity with related isoforms.

What protocols yield optimal results for detecting CAR2 in ocular tissues?

Optimized protocols for CAR2 detection in ocular tissues include:

• Tissue preparation considerations:

  • Fix tissues in 4% paraformaldehyde for no more than 24 hours

  • Utilize cryopreservation when possible to maintain antigen integrity

  • Perform careful orientation during embedding to enable proper sectioning of ciliary body

• Immunohistochemistry protocol:

  • Implement heat-mediated antigen retrieval in citrate buffer (pH 6.0)

  • Block with 5-10% serum from the same species as the secondary antibody

  • Incubate with CAR2 primary antibody at 1:100-1:500 dilution overnight at 4°C

  • Utilize tyramide signal amplification for enhanced sensitivity in paraffin sections

• Immunofluorescence optimizations:

  • Consider optical clearing techniques for whole-mount preparations

  • Implement confocal microscopy with z-stacking for three-dimensional visualization

  • Use DAPI or similar nuclear counterstain for structural context

• Western blot adaptations:

  • Homogenize ocular tissues in RIPA buffer with protease inhibitors

  • Include 1-2% SDS in the lysis buffer to improve extraction efficiency

  • Run samples under reducing conditions with 10-12% polyacrylamide gels

  • Transfer to PVDF membranes at lower voltage overnight for improved transfer
    These specialized protocols significantly improve detection sensitivity and specificity in challenging ocular tissues.

How can CAR2 antibodies facilitate novel therapeutic development for glaucoma?

CAR2 antibodies serve as essential tools in developing glaucoma therapeutics through:

• Target validation:

  • Confirm expression patterns in human and animal model tissues relevant to glaucoma

  • Verify protein reduction following genetic interventions (CRISPR-Cas9)

  • Correlate CAR2 levels with functional outcomes like intraocular pressure reduction

• Mechanism-of-action studies:

  • Assess protein levels following small molecule inhibitor treatment

  • Compare efficacy of gene therapy approaches versus conventional CAIs

  • Evaluate potential compensatory mechanisms through profiling related proteins

• Therapeutic monitoring:

  • Develop custom assays for quantifying target engagement

  • Assess duration of therapeutic effect following single-dose gene therapy

  • Identify potential biomarkers of treatment response

• Translation to clinical applications:

  • Validate findings across species using cross-reactive antibodies

  • Develop predictive assays for patient stratification

  • Establish quality control measures for therapeutic development
    CAR2 antibodies thus provide critical tools throughout the therapeutic development pipeline, from target validation to clinical translation .

What emerging techniques are enhancing the utility of CAR2 antibodies in research?

Several innovative approaches are expanding CAR2 antibody applications:

• Advanced imaging techniques:

  • Super-resolution microscopy for subcellular localization

  • Light sheet microscopy for whole-organ imaging with cellular resolution

  • Expansion microscopy for enhanced visualization of intracellular structures

• Protein-protein interaction studies:

  • Proximity ligation assays to detect CAR2 interactions with regulatory proteins

  • Co-immunoprecipitation coupled with mass spectrometry to identify novel binding partners

  • FRET-based approaches to study dynamic interactions in living cells

• Single-cell analysis:

  • Combining CAR2 antibodies with single-cell RNA sequencing data

  • Mass cytometry (CyTOF) for high-dimensional protein profiling

  • Spatial transcriptomics correlated with CAR2 protein distribution

• Functional genomics integration:

  • Correlating CRISPR screening data with protein expression patterns

  • Validating gene editing outcomes at the protein level

  • Identifying synthetic lethal interactions through combinatorial approaches
    These emerging techniques significantly expand the information obtainable from CAR2 antibody-based studies beyond traditional detection applications.

What are the key factors in selecting the optimal CAR2 antibody for specific research applications?

Selection of the optimal CAR2 antibody should be guided by:

• Application compatibility:

  • Western blot applications: Select antibodies specifically validated for this technique

  • Immunohistochemistry: Choose antibodies tested on relevant fixed tissues

  • Multiple applications: Consider polyclonal antibodies with broader epitope recognition

• Species reactivity requirements:

  • Human-specific studies: Numerous validated options available

  • Cross-species studies: Select antibodies with demonstrated reactivity across target species

  • Evolutionary studies: Consider epitope conservation across phylogeny

• Technical specifications:

  • Conjugation requirements (unconjugated vs. labeled)

  • Clonality (monoclonal for reproducibility, polyclonal for sensitivity)

  • Host species (avoid same species as experimental samples)

  • Validation documentation and published citations

• Experimental design considerations:

  • Multiplexing needs (host species compatibility with other antibodies)

  • Signal amplification requirements

  • Quantitative vs. qualitative analysis goals
    Careful consideration of these factors ensures selection of reagents optimally suited to specific research objectives.

How can researchers contribute to improving CAR2 antibody validation standards?

Researchers can enhance CAR2 antibody validation through:

• Comprehensive validation protocols:

  • Test across multiple applications and biological contexts

  • Include genetic knockout/knockdown controls

  • Verify specificity against related carbonic anhydrase isoforms

• Transparent reporting:

  • Document complete antibody information in publications

  • Share detailed protocols including optimization steps

  • Report negative results and limitations

• Community-based validation:

  • Contribute validation data to antibody validation initiatives

  • Participate in multi-laboratory validation studies

  • Share alternative protocols for challenging applications

• Advanced validation approaches:

  • Implement orthogonal detection methods

  • Use mass spectrometry validation of immunoprecipitated targets

  • Develop quantitative standards for expression analysis These practices collectively improve research reproducibility and accelerate scientific progress in CAR2-related research fields. The continuing development of CAR2 antibody applications, particularly in the context of glaucoma gene therapy and other ocular research, represents a promising frontier in both basic science and therapeutic development. Researchers who implement robust validation and methodological approaches will be best positioned to contribute to this rapidly evolving field.