CA2 Antibody

What is CA2 and why is it an important research target?

Carbonic Anhydrase II (CA2) is a cytosolic enzyme that catalyzes the reversible hydration of carbon dioxide. It represents one of the most widely distributed isoforms among mammalian carbonic anhydrases . CA2 plays critical roles in cellular ion transport, pH regulation, and homeostasis. Its importance extends to bone resorption, osteoclast differentiation, and brain development . Defects in CA2 are associated with significant clinical conditions including osteopetrosis and renal tubular acidosis . Additionally, CA2 shows altered expression in neurological conditions such as Alzheimer's disease and Down syndrome , and it serves as a diagnostic marker for Gastrointestinal Stromal Tumors (GISTs) .

How do I select the appropriate CA2 antibody for my experiment?

Selecting the optimal CA2 antibody requires consideration of several factors:

• Target species compatibility: Available CA2 antibodies show reactivity with various species including human, mouse, and rat samples . Ensure the antibody is validated for your species of interest.

• Application suitability: Verify the antibody has been validated for your specific application:

  • Western Blot (WB): Most common application, typically at dilutions of 1:500-1:2000

  • Immunoprecipitation (IP): Generally requires 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Immunohistochemistry (IHC): Often requires optimization for specific tissue types

  • ELISA: Available for quantitative analyses

• Antibody format: Choose between:

  • Monoclonal antibodies: Greater specificity for a single epitope (e.g., MAB2184)

  • Polyclonal antibodies: Broader epitope recognition (e.g., AF2184)

• Host species: Consider downstream applications to avoid cross-reactivity (options include rabbit , rat , and sheep anti-CA2 antibodies)

What are the critical steps for validating a CA2 antibody?

Proper antibody validation is essential for research rigor and reproducibility :

For Western blot validation specifically:

• Run a representative full blot showing specificity for CA2 at its expected molecular weight (~27-29 kDa)

• Include positive controls like human kidney tissue, which consistently shows strong CA2 expression

• Document nonspecific bands and explain their significance

What are the optimal protocols for CA2 detection by Western blot?

For successful Western blot detection of CA2:

• Protein loading:

  • For tissue lysates (heart, kidney, colon): Typically load 10-20 μg total protein

  • For cell lines (Caki-2, A431, HEK-293): 20-30 μg protein is often sufficient

• Sample preparation:

  • Use reducing conditions as demonstrated in validated protocols

  • CA2 is detected at approximately 27-29 kDa

• Primary antibody incubation:

  • For polyclonal antibodies (e.g., AF2184): Use 1 μg/mL concentration

  • For monoclonal antibodies (e.g., MAB2184): Use 2 μg/mL concentration

  • Optimize antibody dilution based on signal strength (typically 1:500-1:2000)

• Detection systems:

  • HRP-conjugated secondary antibodies work effectively (e.g., HAF016 for sheep primaries, HAF005 for rat primaries)

  • Consider using specialized buffer systems like Immunoblot Buffer Group 1 for optimal results

How should I optimize immunohistochemistry protocols for CA2 detection?

For effective IHC detection of CA2:

• Tissue processing and fixation:

  • Paraffin-embedded sections work well with CA2 antibodies following antigen retrieval

  • Immersion fixation has been validated for tissues such as human descending colon and ileum

• Antibody concentration and incubation:

  • For monoclonal antibodies (e.g., MAB2184): 0.5 μg/mL overnight at 4°C

  • Primary antibody concentration may require titration for different tissue types

• Detection systems:

  • Fluorescent conjugated secondary antibodies (e.g., NorthernLights 557-conjugated Anti-Rat IgG) provide good visualization

  • Counterstaining with DAPI helps visualize cellular context

• Controls:

  • Include tissue sections known to express CA2 (e.g., kidney, colon) as positive controls

  • Always run a no-primary antibody control to assess background staining

What are the key considerations for immunoprecipitation using CA2 antibodies?

For successful immunoprecipitation of CA2:

• Antibody amount:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Specific antibodies like CA2 Antibody (Catalog #8612) have been validated for IP at 1:50 dilution

• Sample preparation:

  • Mouse kidney tissue has been validated as a reliable source for CA2 immunoprecipitation

• Technical considerations:

  • Pre-clear lysates to reduce nonspecific binding

  • Include appropriate negative controls (non-immune IgG from the same species as the antibody)

  • Verify IP results with Western blot to confirm specificity

How can I use CA2 antibodies to study disease mechanisms?

CA2 antibodies have been essential in elucidating disease mechanisms:

• Neurological disorders:

  • CA2 shows elevated expression in Alzheimer's disease patients

  • CA2 expression analysis in developing brains of Down syndrome patients reveals altered patterns

• Gastrointestinal pathologies:

  • CA2 serves as a diagnostic marker for Gastrointestinal Stromal Tumors (GISTs)

  • Immunohistochemical analysis of CA2 in human descending colon and ileum provides insights into digestive system disorders

• Skeletal disorders:

  • CA2 is essential for bone resorption and osteoclast differentiation

  • Defects in CA2 are associated with osteopetrosis, making antibody detection crucial in understanding disease progression

Methodological approach: Use dual antibody labeling with markers for specific cell types to understand cell-specific expression changes in diseased tissues.

What approaches can resolve contradictory results when using different CA2 antibodies?

When faced with contradictory results:

• Compare antibody epitopes:

  • Different antibodies may target different regions of CA2

  • Polyclonal antibodies recognize multiple epitopes, while monoclonal antibodies target specific epitopes

  • Check if the epitope is conserved across species if working with non-human samples

• Validation with multiple techniques:

  • Confirm results using complementary techniques (e.g., if WB shows unexpected results, verify with IHC or IP)

  • Consider genetic approaches (siRNA knockdown) to validate antibody specificity

• Cross-validation with multiple antibodies:

  • Use antibodies from different sources targeting different epitopes

  • Compare results from both monoclonal (e.g., MAB2184) and polyclonal (e.g., AF2184) antibodies

• Protein complex considerations:

  • CA2 interacts with monocarboxylate transporter isoform 1 and 4 (MCT1/4), which may affect epitope accessibility

  • Consider using detergents that preserve or disrupt protein-protein interactions

How can I quantitatively assess CA2 expression levels across different experimental conditions?

For quantitative CA2 expression analysis:

• Western blot densitometry:

  • Ensure linear range of detection by testing multiple exposure times

  • Normalize to appropriate loading controls

  • Use technical replicates across multiple blots to account for gel-to-gel variation

• ELISA-based quantification:

  • Several CA2 antibodies are validated for ELISA applications

  • Develop standard curves using recombinant CA2 proteins for absolute quantification

• Simple Western™ analysis:

  • This capillary-based immunoassay provides more precise quantification

  • Has been validated for CA2 detection at approximately 37 kDa in tissue lysates loaded at 0.2 mg/mL

• Image analysis for IHC:

  • Use digital image analysis software to quantify staining intensity

  • Establish clear thresholds for positive staining

  • Analyze multiple fields per section and multiple sections per sample

What are the latest innovative applications of CA2 antibodies in research?

Recent innovative applications include:

• Protein-protein interaction studies:

  • CA2 has been identified as facilitating transporter activity of MCT1/4 independent of its catalytic activity

  • Co-immunoprecipitation with CA2 antibodies can help identify novel binding partners

• Multi-omics integration:

  • Combining CA2 immunodetection with transcriptomics data provides insights into post-transcriptional regulation

  • Example: Transcriptomic atlas studies using CA2 antibodies revealed cell-specific expression patterns in CNS demyelination and remyelination

• Therapeutic target validation:

  • CA2 antibodies are used to validate the efficacy of carbonic anhydrase inhibitors

  • Monitor CA2 expression changes in response to therapeutic interventions

• High-throughput screening applications:

  • CA2 antibodies can be incorporated into microarray-based assays for drug screening

  • Automated image analysis of CA2 immunofluorescence enables large-scale phenotypic screening

What are common issues encountered when using CA2 antibodies and how can they be resolved?

How can I determine the optimal antibody concentration for my specific experimental conditions?

For optimal antibody titration:

• Western blot optimization:

  • Test serial dilutions (e.g., 1:500, 1:1000, 1:2000)

  • Assess signal-to-noise ratio at each concentration

  • Select dilution that gives strong specific signal with minimal background

• Immunohistochemistry optimization:

  • Begin with manufacturer's recommended concentration (e.g., 0.5 μg/mL for MAB2184)

  • Perform serial dilutions on known positive control tissues

  • Evaluate specific staining versus background at each concentration

• General approach:

  • "Optimal dilutions should be determined by each laboratory for each application"

  • Maintain detailed records of optimization experiments

  • Consider tissue-specific or cell line-specific optimization

What quality control measures should be implemented when using CA2 antibodies long-term?

For consistent long-term results:

• Antibody storage and handling:

  • Store according to manufacturer recommendations (typically at -20°C)

  • Avoid repeated freeze-thaw cycles

  • Consider aliquoting antibodies for single use

• Lot testing and validation:

  • Test new antibody lots against previous lots

  • Maintain reference samples as positive controls

  • Document lot numbers and validation results

• Regular control testing:

  • Include positive and negative controls in each experiment

  • Monitor control results over time for consistency

  • Establish acceptance criteria for control performance

• Protocol standardization:

  • Maintain detailed standard operating procedures

  • Use consistent reagents and instruments

  • Implement quality control checkpoints throughout protocols