CAR4 Antibody

What is Carbonic Anhydrase IV (CAR4/CA4) and why is it studied?

Carbonic Anhydrase IV (CAR4/CA4) is a membrane-associated enzyme that is anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage . It belongs to the carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. CAR4 is predominantly expressed in specific tissues including kidney tubules and pulmonary capillaries, as evidenced by immunohistochemical staining patterns . Researchers study CAR4 due to its involvement in critical physiological processes such as acid-base balance, CO2 transport, and ion exchange in various tissues. Understanding CAR4 function and expression patterns provides valuable insights into normal physiology and potential pathological conditions affecting these processes. The membrane-bound nature of CAR4 makes it particularly interesting for researchers investigating cell-surface protein functions and tissue-specific expression patterns.

What types of CAR4 antibodies are available for research purposes?

Researchers have access to both polyclonal and monoclonal antibodies targeting Carbonic Anhydrase IV (CAR4/CA4). Polyclonal antibodies, such as rabbit polyclonal antibodies to CAR4, are produced by immunizing rabbits with purified recombinant human CAR4 protein (NP_000708.1; Met 1-Lys 283) . These antibodies recognize multiple epitopes on the CAR4 protein and are purified through antigen affinity chromatography. Monoclonal antibodies against CAR4 are also available, providing highly specific recognition of CAR4 epitopes with minimal cross-reactivity . For instance, rabbit monoclonal antibodies to CAR4 have been validated to show no cross-reactivity with other human carbonic anhydrase family members including CA2, CA3, CA5A, CA5B, CA8, CA9, CA10, CA12, CA13, and CA14 in ELISA testing . This specificity makes monoclonal antibodies particularly valuable for applications requiring precise target recognition. Both antibody types are typically preserved in PBS with trehalose and can be stored at 2-8°C for short-term use or at -20°C to -80°C for long-term stability .

How can I determine if my CAR4 antibody is suitable for my specific application?

Determining antibody suitability requires evaluating several key characteristics of the antibody in relation to your experimental needs. First, review the validated applications listed in the antibody datasheet, which typically include Western blot (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), immunoprecipitation (IP), or ELISA, along with the recommended working concentrations for each application . Second, examine the species reactivity information to ensure the antibody will recognize your target protein from the species you're studying; for example, many CAR4 antibodies are specifically validated for human CAR4/CA4 . Third, assess the antibody's specificity by reviewing cross-reactivity data, particularly important when studying a protein belonging to a family with high sequence homology like carbonic anhydrases . Fourth, examine validation images provided by the manufacturer showing actual experimental results, such as IHC images of CAR4 staining in kidney and lung tissues, which can give you confidence in the antibody's performance . Finally, consider conducting preliminary validation experiments in your own laboratory using positive control samples known to express CAR4 and negative controls where the protein is absent to confirm the antibody's performance in your specific experimental system.

What are the optimal conditions for using CAR4 antibodies in immunohistochemistry (IHC)?

When performing immunohistochemistry with CAR4 antibodies, several critical parameters must be optimized for successful detection. First, tissue fixation and processing significantly impact antibody performance, with formalin-fixed paraffin-embedded (FFPE) sections being the validated format for most CAR4 antibodies . For antigen retrieval, heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically recommended to expose antigenic sites that may have been masked during fixation. Antibody concentration should be carefully titrated, with polyclonal antibodies typically used at 0.5-2 μg/mL and monoclonal antibodies at 1-10 μg/mL for IHC-P applications . Incubation conditions also greatly influence staining quality, with overnight incubation at 4°C often yielding better signal-to-noise ratios than shorter incubations at room temperature. For detection systems, both chromogenic (DAB) and fluorescent secondary antibodies are compatible with CAR4 detection, though chromogenic methods are more commonly used for visualizing CAR4 in tissues such as kidney tubules and pulmonary capillaries . Including appropriate positive control tissues (kidney and lung) and negative controls (isotype control antibodies or tissues known not to express CAR4) is essential for validating staining specificity and troubleshooting any technical issues.

How should I optimize Western blot protocols for detecting CAR4 protein?

Optimizing Western blot protocols for CAR4 detection requires attention to several key variables that influence detection sensitivity and specificity. Sample preparation is critical, with membrane proteins like CAR4 requiring appropriate extraction buffers containing mild detergents (such as 1% Triton X-100 or 0.5% NP-40) to solubilize the protein while preserving its native conformation. For gel electrophoresis, using 10-12% polyacrylamide gels typically provides optimal resolution for CAR4, which has a molecular weight of approximately 35-40 kDa. During protein transfer to membranes, PVDF membranes often provide better results than nitrocellulose for detecting membrane-associated proteins like CAR4, with transfer conditions of 100V for 1-2 hours in cold transfer buffer containing 20% methanol. Blocking solutions should be optimized, with 5% non-fat dry milk in TBST usually working well for polyclonal antibodies, while 3-5% BSA in TBST may provide lower background for some monoclonal antibodies . Primary antibody concentration is typically used at 10-20 μg/mL for Western blot applications, with overnight incubation at 4°C to maximize binding specificity . Signal detection can be accomplished using either chemiluminescence or fluorescent secondary antibodies, with the latter offering advantages for quantitative analysis. For troubleshooting weak signals, extending primary antibody incubation time, increasing antibody concentration, or using signal enhancement systems might improve detection sensitivity.

What considerations are important when using CAR4 antibodies for immunoprecipitation (IP)?

Immunoprecipitation with CAR4 antibodies requires specific considerations due to CAR4's nature as a membrane-anchored protein. First, cell lysis buffers must be carefully selected to efficiently solubilize CAR4 while preserving antibody-binding epitopes; RIPA buffer containing 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS is often effective for membrane proteins. The recommended antibody concentration for IP applications is typically 4-8 μg per mg of lysate , though this should be optimized for each specific experimental system. Pre-clearing the lysate with protein A/G beads before adding the CAR4 antibody can significantly reduce non-specific binding and background. Incubation conditions are critical, with overnight incubation at 4°C with gentle rotation generally yielding optimal antibody-antigen binding. For bead selection, protein A sepharose beads work well for rabbit IgG antibodies commonly used for CAR4 detection . Multiple gentle washing steps with decreasing stringency buffers help minimize background while preserving specific interactions. When eluting the immunoprecipitated complexes, using non-reducing conditions may better preserve the native conformation of CAR4 for downstream applications. Including appropriate controls is essential: an isotype control antibody helps identify non-specific binding, while input samples (pre-IP lysate) confirm the presence of target protein prior to enrichment. For verification of successful immunoprecipitation, Western blotting using a different CAR4 antibody recognizing a distinct epitope provides the most convincing evidence of specificity.

How can I distinguish between CAR4 and other carbonic anhydrase isoforms in my experiments?

Distinguishing between CAR4 and other carbonic anhydrase isoforms requires a multifaceted approach due to the high sequence homology within this enzyme family. First, select antibodies with validated specificity for CAR4, such as monoclonal antibodies that have been tested against multiple carbonic anhydrase isoforms and shown no cross-reactivity with CA2, CA3, CA5A, CA5B, CA8, CA9, CA10, CA12, CA13, and CA14 . Second, incorporate experimental controls using tissues or cell lines with known expression patterns of different carbonic anhydrase isoforms; for example, CAR4 shows distinctive localization to renal tubules and pulmonary capillaries . Third, complement antibody-based detection with mRNA analysis techniques such as RT-qPCR using isoform-specific primers to confirm expression at the transcriptional level. Fourth, consider subcellular localization as a distinguishing characteristic - CAR4 is membrane-anchored via a phosphatidylinositol glycan linkage, while other isoforms may be cytosolic or have different membrane associations . Fifth, functional assays targeting carbonic anhydrase activity can be performed with isoform-selective inhibitors to further differentiate between family members. Finally, mass spectrometry-based approaches can provide definitive identification of carbonic anhydrase isoforms by detecting isoform-specific peptide sequences, particularly valuable in complex samples where multiple isoforms may be present simultaneously.

What are common pitfalls when analyzing CAR4 expression in tissue samples, and how can they be avoided?

When analyzing CAR4 expression in tissue samples, researchers frequently encounter several pitfalls that can compromise data interpretation. First, fixation artifacts can significantly alter CAR4 epitope accessibility; standardizing fixation protocols (preferably using 10% neutral buffered formalin for 24-48 hours) and validating antibody performance in similarly fixed positive control tissues helps mitigate this issue . Second, non-specific binding can lead to false-positive results, particularly with polyclonal antibodies; this can be minimized by optimizing blocking conditions, antibody concentration, and implementing appropriate negative controls such as isotype antibodies or CAR4-negative tissues. Third, autofluorescence in tissues like lung (where CAR4 is expressed in pulmonary capillaries) can interfere with immunofluorescence detection; employing autofluorescence quenching reagents or using chromogenic detection methods can overcome this challenge . Fourth, misinterpretation of cellular localization can occur due to CAR4's membrane association; high-resolution imaging and co-staining with membrane markers can help accurately determine subcellular distribution. Fifth, variations in CAR4 expression across different tissue preparations or patient samples may be misattributed to biological differences when they actually result from technical variations; including internal reference proteins and standardizing all aspects of sample handling help ensure comparability. Finally, antibody lot-to-lot variations can introduce inconsistencies in longitudinal studies; validating each new antibody lot against previous results using identical positive control samples maintains data integrity across experiments.

What strategies can improve detection sensitivity for low-expressing CAR4 in challenging samples?

Detecting low-expressing CAR4 in challenging samples requires specialized approaches to enhance sensitivity while maintaining specificity. First, implement signal amplification methods such as tyramide signal amplification (TSA) for immunohistochemistry or chemiluminescence substrates with extended reaction times for Western blotting, which can increase detection sensitivity by 10-100 fold compared to conventional methods. Second, optimize sample preparation to concentrate CAR4 protein; for membrane-bound proteins like CAR4, using membrane fractionation techniques prior to analysis can significantly enrich target concentration relative to whole cell lysates. Third, employ more sensitive detection systems such as highly-sensitive ELISA assays, which can detect CAR4 at levels as low as 0.039 ng/well according to validated antibody specifications . Fourth, consider using indirect immunofluorescence with photomultiplier tube-based detection or electron microscopy with gold-conjugated secondary antibodies for visualization at the ultrastructural level. Fifth, implement epitope retrieval optimization by testing multiple buffers and conditions systematically, as enhanced retrieval can dramatically improve antibody accessibility to CAR4 epitopes, particularly in fixed tissues. Finally, consider using multiple CAR4 antibodies recognizing different epitopes in parallel experiments to confirm findings, as detection challenges may be epitope-specific rather than reflecting true absence of the protein. With particularly challenging samples, combining protein detection with mRNA analysis methods like RNAscope or in situ hybridization can provide complementary evidence of CAR4 expression.

How should quantitative differences in CAR4 expression be analyzed across different experimental conditions?

Analyzing quantitative differences in CAR4 expression requires rigorous methodological approaches to ensure valid comparisons across experimental conditions. First, establish standardized quantification methods appropriate to your detection technique: for Western blots, densitometry with normalization to loading controls (e.g., β-actin, GAPDH) or membrane proteins (e.g., Na⁺/K⁺-ATPase) for membrane-bound CAR4; for IHC, consider digital image analysis with consistent acquisition parameters and either H-score, Allred scoring, or automated pixel intensity quantification . Second, implement adequate biological and technical replicates (minimum three biological and two technical replicates) to enable statistical analysis and account for natural variation. Third, include calibration standards when possible, such as recombinant CAR4 protein at known concentrations for Western blots or ELISA, establishing a standard curve for absolute quantification. Fourth, employ appropriate statistical tests based on your experimental design and data distribution: parametric tests (t-test, ANOVA) for normally distributed data or non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) when normality cannot be assumed. Fifth, consider the dynamic range limitations of your detection method, particularly for highly abundant or very low-expressing samples, and adjust exposure times or antibody concentrations accordingly to ensure measurements fall within the linear range of detection. Finally, when comparing CAR4 expression across different tissues or cell types, account for cell-type specific differences in reference gene expression that might skew normalization; in such cases, multiple reference genes or absolute quantification methods may provide more reliable comparisons.

What are the most reliable markers to correlate with CAR4 expression in functional studies?

When conducting functional studies involving CAR4, correlating its expression with appropriate markers provides valuable context for interpreting results. First, consider pH-sensitive fluorescent probes (such as BCECF or pHrodo) as functional correlates, since CAR4's enzymatic activity directly influences local pH regulation through carbonic acid hydration/dehydration. Second, incorporate membrane markers like caveolin-1 or flotillin-1 to correlate with CAR4's GPI-anchored membrane localization, providing insights into potential functional microdomains where CAR4 operates . Third, analyze ion transporters functionally linked to carbonic anhydrases, such as chloride/bicarbonate exchangers (AE1, AE2), sodium/bicarbonate cotransporters (NBCs), or sodium/proton exchangers (NHEs), which often work in concert with CAR4 in pH and ion homeostasis. Fourth, examine tissue-specific functional markers—in kidney samples, correlate CAR4 with markers of specific tubular segments (proximal tubule: megalin, cubilin; distal tubule: thiazide-sensitive NaCl cotransporter) to contextualize its role in renal physiology . Fifth, assess vascular markers in lung tissue (CD31, VE-cadherin) to correlate with CAR4's expression in pulmonary capillaries, providing insights into its potential roles in pulmonary gas exchange . Finally, consider functional readouts related to carbonic anhydrase activity, such as bicarbonate transport rates or CO2 hydration kinetics measured using stopped-flow spectrophotometry, which can directly link CAR4 expression levels to enzymatic activity in experimental systems.

How can I differentiate technical artifacts from true biological variability in CAR4 antibody-based experiments?

Distinguishing technical artifacts from true biological variability in CAR4 antibody experiments requires systematic troubleshooting and appropriate controls. First, implement experimental design that includes both biological replicates (different samples) and technical replicates (same sample processed multiple times) to quantify variation at different levels; consistent results across technical replicates but differences between biological replicates suggest true biological variability. Second, include procedural controls targeting each step of your protocol: for Western blots, use positive control lysates with known CAR4 expression; for IHC, include established CAR4-positive tissues (kidney, lung) processed in parallel with experimental samples . Third, employ antibody validation controls including: (a) preabsorption controls where primary antibody is pre-incubated with recombinant CAR4 protein before application, which should eliminate specific staining; (b) secondary-only controls to detect non-specific binding of detection reagents; and (c) isotype controls using non-specific IgG from the same species as the primary antibody. Fourth, verify results with orthogonal methods that don't rely on antibodies, such as in situ hybridization for CAR4 mRNA or mass spectrometry-based protein identification. Fifth, implement batch controls where samples from different experimental conditions are processed simultaneously using the same reagent preparations to minimize technical variables. Finally, unexpected subcellular localization or molecular weight variations often indicate technical issues; CAR4 should consistently show membrane localization in IHC and a specific molecular weight (35-40 kDa) in Western blots based on validated results .

How can CAR4 antibodies be utilized in studying interactome networks of membrane-associated proteins?

CAR4 antibodies offer powerful tools for elucidating the interactome networks of this membrane-associated protein through several advanced approaches. First, co-immunoprecipitation (co-IP) using anti-CAR4 antibodies allows for the isolation of protein complexes containing CAR4, which can then be identified through mass spectrometry analysis; this technique is particularly effective when using the recommended 4-8 μg of antibody per mg of lysate with appropriate detergent conditions that preserve protein-protein interactions . Second, proximity labeling methods can be implemented by conjugating CAR4 antibodies to enzymes like BioID or APEX2, enabling the biotinylation of proteins in close proximity to CAR4 in living cells, followed by streptavidin pull-down and mass spectrometry identification. Third, in situ proximity ligation assay (PLA) using CAR4 antibodies paired with antibodies against suspected interaction partners can visualize and quantify specific protein interactions at their native cellular locations with high sensitivity. Fourth, for membrane microdomain studies, CAR4 antibodies can be employed in detergent-resistant membrane fraction analysis to investigate its association with lipid rafts, relevant due to CAR4's GPI-anchor . Fifth, super-resolution microscopy techniques such as STORM or PALM using fluorophore-conjugated CAR4 antibodies can reveal nanoscale co-localization with other membrane proteins that might suggest functional interactions. Finally, competitive binding assays where different proteins compete for binding to CAR4 can be monitored using labeled CAR4 antibodies, providing insights into the dynamics and hierarchy of interaction networks involving this membrane-associated carbonic anhydrase.

What are the considerations for developing CAR-T cell therapies targeting CAR4-expressing tissues?

Developing CAR-T cell therapies targeting CAR4-expressing tissues requires careful consideration of several critical factors. First, it's essential to recognize the fundamental distinction between CAR4 (Carbonic Anhydrase IV) and the CAR (Chimeric Antigen Receptor) technology used in immunotherapy; while both share a similar acronym, they represent entirely different biological entities . Second, when considering CAR4 as a potential target for CAR-T therapy, extensive tissue expression profiling is crucial to understand potential on-target/off-tumor effects, given CAR4's known expression in kidney tubules and pulmonary capillaries . Third, antibody selection for CAR development requires identifying antibodies with high specificity and affinity for CAR4, such as the monoclonal antibodies that demonstrate no cross-reactivity with other carbonic anhydrase family members . Fourth, the design of CAR constructs targeting CAR4 would need to incorporate appropriate single-chain variable fragments (scFvs) derived from these antibodies, connected by either G4S or Whitlow/218 linker sequences between variable heavy and light chains, similar to other CAR designs . Fifth, functional avidity testing of the CAR-T cells against CAR4-expressing target cells would be essential to ensure efficacy while minimizing potential toxicity to healthy tissues expressing CAR4. Finally, preclinical models that accurately recapitulate human CAR4 expression patterns would be needed to evaluate safety and efficacy before clinical translation, similar to the approach used for HBV-targeting CARs where murine models were used to assess on-target activity and potential hepatotoxicity .

How can single-cell analysis techniques be integrated with CAR4 antibody detection for tissue heterogeneity studies?

Integrating single-cell analysis techniques with CAR4 antibody detection enables sophisticated investigations of tissue heterogeneity and cell-specific expression patterns. First, single-cell flow cytometry using fluorescently labeled CAR4 antibodies can quantify expression levels across individual cells within heterogeneous populations, providing distribution patterns rather than population averages; this approach requires optimization of cell isolation protocols that preserve the membrane-bound CAR4 protein . Second, mass cytometry (CyTOF) using metal-conjugated CAR4 antibodies allows simultaneous detection of dozens of additional protein markers, enabling comprehensive phenotyping of CAR4-expressing cells and their molecular signatures without the constraints of fluorescence spectral overlap. Third, imaging mass cytometry or multiplexed ion beam imaging (MIBI) can be employed with CAR4 antibodies to visualize and quantify expression at the single-cell level while maintaining spatial context within tissues, particularly valuable for examining heterogeneity in CAR4-expressing kidney tubules or lung capillaries . Fourth, single-cell sorting based on CAR4 expression followed by transcriptomic analysis (scRNA-seq) can reveal molecular programs associated with different levels of CAR4 expression across individual cells. Fifth, spatial transcriptomics approaches combined with CAR4 immunofluorescence can correlate protein expression with transcriptional profiles in situ, providing insights into regulatory mechanisms within the native tissue architecture. Finally, computational integration of these multimodal single-cell data requires specialized algorithms for dimensionality reduction, clustering, and trajectory analysis to fully characterize the heterogeneity of CAR4-expressing cell populations and their functional implications in both normal physiology and disease states.

What emerging technologies are enhancing CAR4 antibody applications in research?

Emerging technologies are substantially expanding the capabilities and applications of CAR4 antibodies in cutting-edge research. First, CRISPR-mediated genome editing combined with CAR4 antibody detection is enabling precise correlation between genetic modifications and resulting protein expression changes, offering unprecedented insights into the regulatory mechanisms controlling CAR4 expression and function. Second, microfluidic antibody capture technologies are enhancing sensitivity for detecting CAR4 in limited samples such as biopsies or rare cell populations, concentrating the target protein through continuous flow chambers coated with CAR4 antibodies. Third, advanced protein engineering techniques are producing recombinant antibody fragments like nanobodies or single-domain antibodies against CAR4, offering superior tissue penetration and reduced immunogenicity for in vivo imaging applications. Fourth, spatially-resolved proteomics using methods like Digital Spatial Profiling (DSP) with CAR4 antibodies allows multiplexed protein quantification while preserving spatial context within heterogeneous tissues such as kidney or lung . Fifth, artificial intelligence-driven image analysis is revolutionizing the quantification and pattern recognition of CAR4 immunostaining across large tissue datasets, enabling more objective and comprehensive evaluation of expression patterns. Finally, antibody-drug conjugates utilizing CAR4 antibodies are being explored for targeted delivery of therapeutics or imaging agents to CAR4-expressing tissues, leveraging the tissue-specific expression patterns in kidney tubules and pulmonary capillaries observed in validation studies .