CASR Antibody

What is the Calcium-Sensing Receptor (CASR) and why is it significant in research?

CASR is a G-protein-coupled receptor that senses fluctuations in extracellular calcium concentration and plays a key role in maintaining calcium homeostasis. It functions through a G-protein that activates a phosphatidylinositol-calcium second messenger system. CASR is primarily expressed in parathyroid glands and kidneys where it modulates parathyroid hormone secretion and renal calcium reabsorption, respectively. Research significance extends to its role in bone health, muscle contraction, nerve function, and potential involvement in various disorders including hypocalcemia, hypercalcemia, and autoimmune conditions . CASR also demonstrates expression in other tissues including intestine, placenta, and brain, indicating diverse physiological roles beyond calcium regulation .

What applications are CASR antibodies commonly used for in research?

CASR antibodies are utilized across multiple experimental applications:

Researchers should confirm tissue/species reactivity and optimize dilutions for specific experimental conditions .

What are the key differences between monoclonal and polyclonal CASR antibodies?

The choice between monoclonal and polyclonal CASR antibodies depends on research requirements:

Monoclonal antibodies:

• Recognize single epitopes with high specificity

• Examples include CaSR Antibody (6D4) and CaSR (D6D9V) Rabbit mAb

• Provide consistent lot-to-lot reproducibility

• Often preferred for applications requiring high specificity

• May show limited species cross-reactivity depending on epitope conservation

Polyclonal antibodies:

• Recognize multiple epitopes on the CASR protein

• Examples include products like 19125-1-AP and ACR-004

• Potentially higher sensitivity due to binding multiple epitopes

• May show broader species cross-reactivity

• Batch-to-batch variation can be a consideration

What epitope considerations are important when selecting a CASR antibody?

CASR contains distinct domains with different functional significance. Key epitope considerations include:

• Extracellular domain targeting: Antibodies targeting regions aa 41-69, 114-126, 171-195, and 260-340 in the extracellular domain are commonly used. Epitopes in this region can affect receptor activity; for example, autoantibodies recognizing CaSR epitopes 114-126 and 171-195 have been shown to affect receptor activity by increasing inositol-phosphate accumulation and reducing PTH secretion .

• Specific epitope examples:

  • ACR-004 targets aa 216-229 of human CASR (extracellular)

  • MA1-934 targets aa 214-235 of human CASR

  • Some antibodies target the N-terminal region (aa 1-100)

• Functional considerations: The extracellular domain contains multiple glycosylation sites and regions responsible for ligand binding, while transmembrane domains are involved in signaling .

When selecting antibodies, researchers should consider which domain is most relevant to their specific research question .

How can CASR antibody specificity be validated in experimental settings?

Rigorous validation is essential to ensure experimental reliability:

• Knockout/knockdown controls:

  • Use CASR knockout/knockdown cells as negative controls

  • Several publications utilize this approach for validation

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (e.g., Calcium Sensing Receptor Blocking Peptide)

  • Signal should be significantly reduced in competition experiments

• Cross-validation methods:

  • Compare results using multiple antibodies targeting different epitopes

  • Use different detection techniques (WB, IF, IHC) to confirm findings

  • Validate across different cell lines/tissues with known CASR expression

• Recombinant expression systems:

  • Test antibody in HEK293 cells transfected with human CASR versus control transfectants

  • Flow cytometry can confirm surface expression

• Species cross-reactivity testing:

  • Confirm reactivity in target species (human, mouse, rat, etc.)

  • Note that CASR shares ~96% amino acid sequence identity between human and mouse/rat

What are the major challenges in detecting CASR using antibodies and how can they be addressed?

Researchers face several technical challenges when working with CASR antibodies:

• Molecular weight variations:

  • CASR typically appears at ~120-155 kDa in Western blots

  • Glycosylation can affect apparent molecular weight

  • Dimer formation may result in bands at ~260 kDa

  Solution: Include appropriate molecular weight markers and positive controls; consider deglycosylation treatments if necessary.

• Isoform detection:

  • Different antibodies may detect specific CASR isoforms

  • Carefully review antibody documentation for isoform specificity

  Solution: Select antibodies with documented reactivity to your isoform of interest.

• Low endogenous expression:

  • Some tissues express low levels of CASR

  • May require signal amplification strategies

  Solution: Optimize protein loading; consider signal enhancement systems; use tissues with known high expression (parathyroid, kidney) as positive controls.

• Post-translational modifications:

  • Glycosylation and other modifications may affect antibody binding

  Solution: Consider domain-specific antibodies that target regions less affected by modifications.

How can CASR antibodies be utilized to study autoimmune conditions affecting calcium homeostasis?

CASR autoantibodies have been implicated in autoimmune polyendocrine syndrome type 1 (APS1) and other conditions:

• Characterization approaches:

  • Phage-display technology can identify binding sites of autoantibodies

  • ELISA assays using purified CASR can detect autoantibodies in patient sera

  • Functional bioassays measuring inositol-phosphate accumulation or PTH secretion can determine if autoantibodies affect receptor activity

• Key findings from research:

  • APS1 patient autoantibodies target epitopes at aa 41-69, 114-126, 171-195, and 260-340

  • Only autoantibodies against specific epitopes (114-126 and 171-195) affect receptor function

  • Most autoantibodies belong to IgG1 subclass, with a minority of IgG3

• Methodological considerations:

  • Use both detection and functional assays to fully characterize autoantibodies

  • Include healthy control samples for comparison

  • Consider epitope mapping to identify specific binding regions

What role can CASR antibodies play in understanding calcium-related pathologies beyond autoimmune conditions?

CASR antibodies are valuable tools for investigating various calcium-related disorders:

• Hypertension research:

  • CASR and TRPC6 upregulation in pulmonary artery smooth muscle cells has been implicated in idiopathic pulmonary arterial hypertension

  • Antibodies can help track expression changes and localization in disease models

• Genetic disorders:

  • Mutations in CASR can lead to familial hypocalciuric hypercalcemia and neonatal severe primary hyperparathyroidism

  • Immunodetection of mutant receptors can help understand trafficking and expression differences

• Cancer research:

  • CASR expression changes have been observed in various cancers

  • Immunohistochemistry using CASR antibodies can map expression changes in tumor tissues

• Neurological applications:

  • CASR is expressed in rat hippocampal CA1 region (soma and apical dendrites of pyramidal neurons)

  • Antibodies enable precise localization studies in neural tissues

What are common issues in Western blotting with CASR antibodies and how can they be resolved?

Western blotting for CASR presents several technical challenges:

Recommended dilutions for Western blotting range from 1:500 to 1:10,000 depending on the specific antibody and sample source .

How can immunohistochemistry and immunofluorescence protocols be optimized for CASR detection?

For optimal IHC/IF detection of CASR:

• Fixation considerations:

  • Formalin fixation may mask epitopes; consider epitope retrieval methods

  • Some antibodies work better on frozen sections than FFPE tissues

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval may be necessary for some tissues

• Signal amplification:

  • Consider tyramide signal amplification for low-abundance targets

  • Use of appropriate detection systems (HRP, fluorophores) based on experimental needs

• Controls and validation:

  • Include tissue with known CASR expression as positive control

  • Include secondary-only controls to assess background

  • Consider blocking peptides to confirm specificity

• Counterstaining considerations:

  • Nuclear counterstains like DAPI work well with CASR immunofluorescence

  • For brightfield IHC, hematoxylin counterstaining provides good contrast

How can CASR antibodies be applied in flow cytometry for cell surface expression analysis?

Flow cytometry offers advantages for quantifying CASR surface expression:

• Protocol essentials:

  • Use live intact cells to preserve surface epitopes

  • Stain with primary anti-CASR antibody followed by fluorophore-conjugated secondary antibody

  • Include appropriate isotype controls and unstained cells

• Specific examples from literature:

  • Successfully applied for detection of CASR in human THP-1 monocytic leukemia cells

  • Validated in HEK293 cells transfected with human CASR and eGFP versus irrelevant transfectants

  • Set quadrant markers based on control antibody staining

• Available conjugated antibodies:

  • Several directly conjugated antibodies are available including FITC, PE, and Alexa Fluor conjugates

  • These eliminate the need for secondary antibodies in flow cytometry applications

• Considerations:

  • Cell permeabilization should be avoided when studying surface expression

  • If required, gentle fixation methods should be employed

  • Antibodies targeting extracellular domains are preferred for surface detection

What emerging techniques are being developed for studying CASR function using antibodies?

Several innovative approaches utilize CASR antibodies:

• Live cell imaging:

  • Non-permeabilizing protocols allow visualization of CASR trafficking and internalization

  • Time-lapse microscopy can track receptor dynamics in response to calcium or other ligands

• Proximity ligation assays:

  • Can detect CASR interactions with other proteins in situ

  • Provides spatial resolution of protein-protein interactions beyond co-immunoprecipitation

• Super-resolution microscopy:

  • Techniques like STORM or PALM combined with fluorescent antibodies can visualize CASR distribution at nanoscale resolution

  • Helps determine precise membrane localization and clustering

• Mass cytometry:

  • CyTOF technology using metal-conjugated antibodies allows simultaneous detection of numerous markers

  • Useful for complex phenotyping of CASR-expressing cells in heterogeneous populations

• Antibody-based functional modulation:

  • Certain antibodies may mimic or block the effects of calcium on CASR

  • Can be used to study receptor activation mechanisms