CELSR1 Antibody, HRP conjugated

What is CELSR1 and what cellular functions does it perform?

CELSR1 is a receptor that plays a crucial role in cell/cell signaling during nervous system formation . It belongs to the adhesion G protein-coupled receptor (aGPCR) family and is also known by several other names including CDHF9, FMI2, Cadherin family member 9, and Flamingo homolog 2 (hFmi2) . With a calculated molecular weight of 329 kDa, CELSR1 is a large protein with multiple functional domains . Recent research has shown that CELSR1 has neuroprotective effects in cerebral ischemic injury by reducing cell apoptosis in the peri-infarct cerebral cortex and promoting neurogenesis and angiogenesis, primarily through the Wnt/PKC pathway . This makes it an important target for stroke research, as it has been identified as a susceptibility gene for ischemic stroke .

What are the validated applications for CELSR1 antibodies?

Based on current research literature, CELSR1 antibodies have been validated for several experimental applications:

• Western Blot (WB): CELSR1 antibodies such as ab225889 have been validated for detecting the target protein at approximately 329 kDa in human cell lines including HeLa, HEK-293T, and Jurkat .

• Immunoprecipitation (IP): Antibodies like ab225889 have been confirmed effective for pulling down CELSR1 from human cell lysates .

• ELISA: Antibodies such as 20270-1-AP have demonstrated reactivity with human samples in ELISA applications .

When using HRP-conjugated variants, these applications benefit from direct enzymatic detection without requiring secondary antibodies.

What experimental considerations should be made when using CELSR1 antibodies for Western blotting?

When performing Western blot for CELSR1 detection, researchers should consider:

• Sample preparation: NETN buffer has been validated for lysate preparation . Due to the large size of CELSR1 (329 kDa), complete protein extraction and denaturation are critical.

• Gel selection: Use low percentage gels (4-6%) or gradient gels to effectively resolve high molecular weight proteins.

• Transfer conditions: For large proteins, extend transfer times or use specialized protocols for high molecular weight proteins (wet transfer with low methanol buffers).

• Antibody concentration: An optimal concentration of 0.1 μg/mL has been reported for ab225889 .

• Detection: When using HRP-conjugated antibodies, ECL (enhanced chemiluminescence) technique provides appropriate sensitivity, with reported exposure times of approximately 3 minutes .

How should CELSR1 antibodies be stored to maintain activity?

For optimal maintenance of antibody activity:

• Store CELSR1 antibodies at -20°C .

• Antibodies formulated in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 have demonstrated good stability .

• Antibodies are typically stable for one year after shipment when stored properly .

• For smaller aliquots (e.g., 20μL sizes), BSA may be included (0.1%) as a stabilizer .

• Aliquoting is generally unnecessary for -20°C storage based on manufacturer recommendations .

How can researchers optimize CELSR1 detection in neurogenesis and angiogenesis studies?

For optimizing CELSR1 detection in neurogenesis and angiogenesis studies following cerebral ischemic injury:

• Regional considerations: The mRNA expression of CELSR1 shows differential regulation after ischemia/reperfusion in different brain regions: increased in the subventricular zone (SVZ) and dentate gyrus (DG), decreased in the ischemic penumbra, and relatively unchanged in the ischemic core and striatum . Target tissue collection should consider these regional variations.

• Co-staining protocols: Implement dual or triple immunostaining protocols using:

  • CELSR1 antibody (HRP-conjugated or primary with compatible secondary)

  • Neurogenesis markers (e.g., DCX for neuroblasts)

  • CD31 for vascular endothelial cell detection

• Quantification methods: Quantify CD31-positive vascular area ratio for angiogenesis assessment as demonstrated in previous studies (where control groups showed approximately 24.61% ± 3.68% CD31-positive area ratio around the SVZ) .

• Timing considerations: CELSR1 expression changes dynamically after ischemic injury. Research indicates significant changes after 2 hours of ischemia followed by 22 hours of reperfusion .

What methodology should be employed to investigate CELSR1's G protein coupling mechanism?

To investigate CELSR1's G protein coupling mechanism:

• BRET2 assay implementation: Bioluminescence resonance energy transfer (BRET2) sensors can be used to assay the "transducerome" of G protein combinations, as demonstrated for other adhesion GPCRs like LPHN3 .

• Thrombin-mediated acute exposure approach: This methodology can be adapted to evaluate acute tethered-agonist (TA) dependent G protein coupling of CELSR1 .

• G protein knockout cell models: Conduct experiments in G protein knockout (GKO) HEK293 cells to minimize interference between endogenous G proteins and overexpressed receptors of interest .

• Controls: Include empty vector (EV) controls to identify non-specific effects of treatments such as thrombin .

• Validation: Compare CELSR1 with established aGPCRs like LPHN3 that have known G protein coupling profiles to validate experimental design .

How can researchers detect both cleaved and uncleaved forms of CELSR1?

Based on recent research findings, detection of both cleaved and uncleaved forms of CELSR1 requires specific experimental approaches:

• Dual tagging strategy: Use N- and C-terminal tags (e.g., HA and FLAG respectively) to monitor the putative N- or C-terminal fragments resulting from potential autoproteolytic cleavage .

• Immunoblotting protocol:

  • For CELSR1, be aware that predominant products correspond to uncleaved, full-length receptors (unlike CELSR2 which shows effective cleavage) .

  • Include positive controls such as human LPHN3, which is established to be effectively cleaved .

  • Run appropriate molecular weight markers to distinguish between full-length (~329 kDa) and cleaved fragments.

• Expression systems: Use HEK293T cells for heterologous expression as they have been validated for studying CELSR proteins .

• Detection optimization: Optimize SDS-PAGE conditions (gradient gels, running time, transfer parameters) to resolve both high molecular weight full-length proteins and smaller cleaved fragments.

What are the key considerations for optimizing immunohistochemical detection of CELSR1 in brain tissue?

For immunohistochemical detection of CELSR1 in brain tissue:

• Tissue preparation:

  • Perfusion-fixed tissue provides better antigen preservation

  • Appropriate post-fixation (typically 24-48 hours in 4% PFA)

  • Cryoprotection in sucrose gradients for frozen sections

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Alternatively, try Tris-EDTA buffer (pH 9.0) if citrate buffer gives suboptimal results

• Detection strategy for HRP-conjugated antibodies:

  • Direct detection using DAB (3,3'-diaminobenzidine) substrate

  • Signal amplification using tyramide signal amplification (TSA) for low-abundance targets

• Region-specific optimization:

  • Different brain regions require different antibody concentrations

  • SVZ and DG regions show higher CELSR1 expression after ischemic injury

• Controls:

  • Include CELSR1 knockdown tissue (using lentiviral shRNA approach as validated in previous studies)

  • Include no-primary-antibody controls to assess non-specific binding

What methodological approaches can be used to study CELSR1's role in neuroprotection?

To investigate CELSR1's role in neuroprotection:

• Knockdown approach:

  • CELSR1-shRNA lentivirus with green fluorescent protein (GFP) has been validated for knockdown studies with approximately 50% interference efficiency .

  • Microinjection into lateral ventricle and DG has been successfully implemented .

• Ischemia model implementation:

  • Middle cerebral artery occlusion (MCAO) rat model is an established approach .

  • Typically performed 12 days after lentivirus injection with analysis 3 days post-MCAO .

• Quantitative assessment methods:

  • Infarct volume measurement

  • Neurological deficit scoring

  • Mortality rate calculation (knockdown of CELSR1 has been shown to increase mortality three-fold)

  • Cell apoptosis quantification in the peri-infarct cerebral cortex

  • Neurogenesis assessment (neuroblast proliferation)

  • Angiogenesis measurement (CD31-positive vascular area ratio)

• Molecular pathway investigation:

  • Assessment of p-PKC expression in the SVZ and peri-infarct tissue

  • Investigation of Wnt/PKC pathway components

What are the common issues with CELSR1 detection and their solutions?

Common challenges in CELSR1 detection and their solutions include:

How can researchers validate the specificity of CELSR1 antibodies?

To validate CELSR1 antibody specificity:

• Positive and negative controls:

  • Use cell lines with known CELSR1 expression (HeLa, HEK-293T, Jurkat have been validated)

  • Include knockdown samples (CELSR1-shRNA with validated 50% knockdown efficiency)

• Immunoblotting confirmation:

  • Verify detection at predicted molecular weight (329 kDa)

  • Compare with alternative antibodies targeting different epitopes

• Immunoprecipitation validation:

  • Perform IP followed by Western blot using antibodies targeting different epitopes

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (corresponding to synthetic peptide within Human CELSR1 aa 2750-2850)

  • Signal should be reduced or eliminated in the presence of competing peptide

• Cross-reactivity assessment:

  • Test against related proteins (CELSR2, CELSR3) to ensure specificity

  • Particularly important given the structural similarities within the CELSR family

What emerging applications of CELSR1 antibodies should researchers consider?

Emerging applications for CELSR1 antibodies that researchers should consider include:

• Stroke therapy development:

  • Using CELSR1 antibodies to track therapeutic response in ischemic stroke models

  • Development of targeted therapeutic approaches leveraging CELSR1's neuroprotective effects

• G protein signaling research:

  • Investigation of cleavage-dependent and cleavage-independent aGPCR:G protein coupling mechanisms

  • Exploration of G protein pathways controlled by CELSRs

• Advanced imaging applications:

  • Super-resolution microscopy to visualize CELSR1 distribution in neural progenitor cells

  • Live-cell imaging to track CELSR1 dynamics during neurogenesis

• Single-cell analysis:

  • Use of CELSR1 antibodies in single-cell proteomics

  • Correlation of CELSR1 expression with cell fate decisions in neural stem cells

• Extracellular vesicle research:

  • Detection of CELSR1 in extracellular vesicles as potential biomarkers

  • Analysis of CELSR1's role in intercellular communication via exosomes

These applications will benefit from continued refinement of HRP-conjugated CELSR1 antibodies and development of new detection strategies for this important neurological target.