Phospho-CASR (Thr888) Antibody

What is the Phospho-CASR (Thr888) Antibody and what does it specifically detect?

The Phospho-CASR (Thr888) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of Calcium Sensing Receptor (CASR) only when phosphorylated at threonine 888. This antibody does not recognize non-phosphorylated CASR or other phosphorylation sites, making it valuable for studying specific CASR phosphorylation events . The specificity is achieved through careful purification processes where non-phospho specific antibodies are removed through chromatography using non-phosphopeptides .

What are the standard applications for Phospho-CASR (Thr888) Antibody in research?

The Phospho-CASR (Thr888) Antibody is validated for several research applications:

The antibody is particularly useful for monitoring CASR phosphorylation status in response to various treatments or conditions, especially those affecting PKC activity or calcium signaling pathways .

What is the molecular and functional significance of CASR Thr888 phosphorylation?

CASR Thr888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and plays a crucial role in receptor regulation. Functionally, Thr888 phosphorylation modulates CASR sensitivity to agonists and influences calcium-induced intracellular signaling patterns .

How should I optimize Western blot protocols for Phospho-CASR (Thr888) Antibody?

For optimal Western blot results with Phospho-CASR (Thr888) Antibody:

• Sample preparation:

  • Use freshly prepared lysates whenever possible

  • Include phosphatase inhibitors (e.g., calyculin) in your lysis buffer to prevent dephosphorylation

  • Maintain samples at 4°C during processing

• Protocol optimization:

  • Start with a 1:1000 dilution for initial testing

  • Use 5% BSA (not milk) for blocking and antibody dilution to reduce background

  • Include a positive control (e.g., lysates from cells treated with PKC activators like PMA)

  • Consider using PVDF membranes which may provide better signal for phospho-specific antibodies

• Detection:

  • Expected molecular weight is approximately 120-140 kDa

  • Validate specificity using phosphopeptide competition assays

What controls are essential when using Phospho-CASR (Thr888) Antibody?

To ensure valid and interpretable results with Phospho-CASR (Thr888) Antibody, include the following controls:

• Positive controls:

  • Lysates from cells treated with phorbol ester (PMA), which activates PKC and increases Thr888 phosphorylation

  • Samples with elevated extracellular calcium (2.5-5 mM) which has been shown to increase CASR(T888) phosphorylation

• Negative controls:

  • Samples treated with PKC inhibitors

  • Lysates from cells expressing CASR(T888A) mutant

  • Samples pre-treated with calyculin (a protein phosphatase inhibitor) combined with PMA

• Specificity controls:

  • Antigen competition assay using the phosphopeptide immunogen

  • Non-phosphorylated peptide controls to confirm phospho-specificity

How can I use Phospho-CASR (Thr888) Antibody to study the relationship between calcium sensing and PKC signaling pathways?

To investigate the complex interplay between calcium sensing and PKC signaling:

• Temporal analysis of phosphorylation:

  • Design time-course experiments using calcium ionophores or extracellular calcium elevation (0.5 mM to 2.5-5 mM)

  • Monitor Thr888 phosphorylation at various timepoints (0-60 minutes)

  • Correlate with downstream signaling events using other markers

• Pharmacological manipulation:

  • Use calcimimetics (e.g., NPS R-467) to potentiate CASR activity

  • Apply calcilytics (e.g., NPS-89636) to inhibit receptor function

  • Combine with PKC activators/inhibitors to dissect pathway crosstalk

• Functional correlation:

  • Pair phosphorylation measurements with calcium oscillation imaging

  • Correlate Thr888 phosphorylation status with intracellular calcium dynamics

  • Use the antibody in conjunction with electrophysiology to correlate phosphorylation with channel activity

What experimental approaches can resolve contradictory results when studying CASR phosphorylation in different cell types?

When facing contradictory results across different experimental systems:

• Cell-type specific regulation analysis:

  • Examine baseline PKC isoform expression in your cell types of interest

  • Compare phosphatase activity profiles between systems

  • Measure total CASR expression and membrane localization

• Advanced methodological approaches:

  • Use phosphoproteomics to identify all phosphorylation sites on CASR simultaneously

  • Apply FRET/BRET-based biosensors to monitor conformational changes in real time

  • Employ super-resolution microscopy to visualize phospho-CASR clustering

• Physiological significance assessment:

  • Compare results between recombinant systems and native tissue

  • Correlate phosphorylation with established functional readouts (Ca²⁺ oscillations, ERK activation)

  • Utilize transgenic models with T888A mutations for in vivo validation

What are the most common causes of false negatives when using Phospho-CASR (Thr888) Antibody, and how can I address them?

Common causes of false negatives include:

• Rapid dephosphorylation:

  • Include phosphatase inhibitors in all buffers (calyculin has been specifically shown to block Thr888 dephosphorylation)

  • Process samples rapidly and maintain at 4°C

  • Consider using phosphatase inhibitor cocktails with broad specificity

• Insufficient PKC activation:

  • Verify your PKC stimulation protocol with established PKC substrate phosphorylation

  • Use multiple PKC activators (PMA, bryostatin, etc.)

  • Optimize stimulation time (peak phosphorylation may be transient)

• Technical factors:

  • Ensure antibody is stored properly (-20°C, avoid freeze-thaw cycles)

  • Try longer incubation times and optimize antibody concentration

  • Consider signal amplification systems for low abundance proteins

How can I quantitatively assess CASR phosphorylation dynamics across different experimental conditions?

For rigorous quantitative analysis of CASR phosphorylation:

• Normalization strategies:

  • Always normalize phospho-signal to total CASR expression (requires parallel blots or stripping/reprobing)

  • Include loading controls appropriate for your experimental system

  • Consider using recombinant phosphorylated standards for absolute quantification

• Advanced quantification methods:

  • Use fluorescent secondary antibodies rather than chemiluminescence for broader linear range

  • Employ internal standard curves if comparing across multiple blots

  • Consider phospho-flow cytometry for single-cell analysis of phosphorylation states

• Temporal and spatial resolution:

  • For subcellular localization, combine IF using phospho-CASR antibody with markers for different cellular compartments

  • Use live cell imaging with phospho-specific biosensors for real-time dynamics

  • Consider FLIM-FRET approaches to monitor conformational changes associated with phosphorylation

What are the optimal storage and handling conditions for Phospho-CASR (Thr888) Antibody to maintain its specificity and sensitivity?

For maximum antibody performance and longevity:

• Storage recommendations:

  • Store at -20°C for long-term (up to one year)

  • For frequent use, aliquot and store at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Working solution preparation:

  • Dilute only the amount needed for immediate use

  • Use fresh dilutions for each experiment

  • Prepare dilutions in recommended buffers (typically PBS with 0.5% BSA)

• Quality control measures:

  • Periodically test antibody performance using positive controls

  • Monitor for changes in background or signal intensity over time

  • Consider including standard curves in long-term studies to track antibody performance

How can I use Phospho-CASR (Thr888) Antibody to investigate the co-agonist activation mechanism of CASR?

To explore the complex co-agonist mechanism where aromatic amino acids (Trp/Phe) act with divalent cations (Ca²⁺/Mg²⁺):

• Experimental design strategies:

  • Set up factorial experiments varying both aromatic amino acids and calcium concentrations

  • Monitor Thr888 phosphorylation as a readout of receptor activation

  • Compare phosphorylation patterns induced by different co-agonist combinations

• Structure-function analysis:

  • Use the antibody to assess how mutations in amino acid binding sites affect phosphorylation

  • Combine with conformation-specific antibodies to link phosphorylation with structural changes

  • Correlate phosphorylation status with binding affinities of different co-agonists

• Signaling pathway discrimination:

  • Determine if Thr888 phosphorylation differs when activated by calcium alone versus co-agonist combinations

  • Investigate whether different co-agonists lead to distinct patterns of receptor phosphorylation

  • Map the temporal relationship between co-agonist binding and Thr888 phosphorylation

What methodological approaches can I use to study the relationship between CASR phosphorylation and calcium oscillations in primary cell cultures?

For investigating the mechanistic link between CASR phosphorylation and calcium oscillations:

• Integrated imaging approaches:

  • Combine calcium imaging using ratiometric dyes (Fura-2) with post-fixation phospho-CASR immunostaining

  • Develop correlative live-cell and immunofluorescence protocols to link real-time calcium dynamics with phosphorylation status

  • Use microfluidic systems for precise temporal control of extracellular stimuli

• Genetic manipulation strategies:

  • Express phosphomimetic (T888D) or phospho-null (T888A) CASR mutants in primary cells

  • Use inducible expression systems to control timing of mutant expression

  • Apply CRISPR-Cas9 gene editing to introduce mutations at endogenous loci

• Pharmacological precision tools:

  • Use specific PKC isoform inhibitors to identify which isoforms mediate Thr888 phosphorylation

  • Apply temporal inhibition protocols to determine critical windows for phosphorylation

  • Combine with phosphatase inhibitors (calyculin) to manipulate phosphorylation/dephosphorylation cycles