CRB2 Antibody

What is CRB2 protein and what is its molecular structure?

CRB2 (crumbs cell polarity complex component 2) is a member of the Crumbs protein family. In humans, the canonical protein has 1285 amino acid residues with a predicted molecular weight of 134.3 kDa. The protein has a large extracellular domain with epidermal growth-factor-like and laminin-A globular domains, a single transmembrane domain, and an intracellular C-terminal domain of 37 amino acids . It undergoes post-translational modifications, including N-glycosylation, which can affect its observed molecular weight in experimental conditions .

Where is CRB2 protein expressed in mammalian tissues?

CRB2 is predominantly expressed in the kidney, particularly in glomeruli, podocytes of the glomerular capillary loops, and parietal glomerular epithelial cells . It is also expressed in the retina and brain, where it plays crucial roles in tissue organization and cellular polarity . The CRB2 marker can specifically be used to identify Parietal Epithelial Cells in kidney tissue . Expression has been confirmed in both human and mouse tissues through various detection methods .

What molecular weight bands should I expect when detecting CRB2 by Western blot?

Western blot detection of CRB2 typically reveals multiple bands, including a prominent band slightly under 150 kDa (corresponding to the canonical form) and another band above 260 kDa in tissue lysates . In cell lines, the endogenous protein is commonly detected at 150 kDa, while overexpressed CRB2 fused to tags such as GFP appears at approximately 180 kDa . These variations in molecular weight might reflect different isoforms, post-translational modifications, or protein complexes, particularly in tissue samples where cellular organization is more complex than in cultured cells .

What are the common synonyms and orthologs for CRB2?

Synonyms for CRB2 include crumbs 2, cell polarity complex component, crumbs family member 2, crumbs-like protein 2, and protein crumbs homolog 2 . CRB2 gene orthologs have been reported in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken . The human CRB2 protein shares high similarity with its mouse counterpart, particularly in the cytoplasmic domain, allowing some antibodies to detect both species .

What are the validated applications for CRB2 antibodies?

CRB2 antibodies have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) . For IHC applications, CRB2 antibodies have shown positive detection in mouse brain tissue, while IF applications have successfully detected CRB2 in mouse eye tissue . The appropriate application depends on the specific research question and sample type being studied.

How should I validate the specificity of a CRB2 antibody?

Validation of CRB2 antibody specificity can be performed through several complementary approaches:

• Knockdown experiments: Transfect cells with shRNA targeting CRB2 mRNA and confirm decreased antibody detection. Studies have shown 49-69% reduction in CRB2 protein levels using this approach .

• Overexpression studies: Express CRB2 fused to a tag (e.g., GFP) and confirm co-detection with both CRB2 antibody and tag-specific antibody .

• Peptide competition assay: Pre-incubate the CRB2 antibody with the original antigen fusion protein (typically 0.2 mg/ml) for 1 hour at room temperature before application in Western blot or immunofluorescence to confirm signal specificity .

• Cross-reactivity testing: Test against related proteins (e.g., CRB3) to ensure the antibody does not recognize other family members .

What are the recommended protocols for Western blot detection of CRB2?

For optimal Western blot detection of CRB2:

• Sample preparation: Dissolve proteins in sample buffer containing 2% SDS, 10% glycerol, 700 mM β-mercaptoethanol, 62.5 mM Tris-HCl (pH 6.8), and 0.05% bromophenol blue .

• Electrophoresis: Load samples on SDS-polyacrylamide gels under reducing conditions .

• Transfer: Transfer proteins to PVDF membranes .

• Blocking: Block membranes for 1 hour at room temperature with 2% BSA in Tris-buffered saline containing 0.1% Tween (TBST) .

• Primary antibody: Incubate overnight at 4°C with the CRB2 antibody (typically 5 μg/ml for custom antibodies or following manufacturer's recommendations) .

• Secondary antibody: Incubate with appropriate secondary antibody conjugated to alkaline phosphatase or horseradish peroxidase .

• Development: Develop using NBT/BCIP for alkaline phosphatase or ECL substrate for horseradish peroxidase .

What are the recommended protocols for immunofluorescence detection of CRB2?

For immunofluorescence detection of CRB2 in tissues such as retinal pigment epithelium (RPE):

• Fixation: Fix tissues in 4% paraformaldehyde for 10 minutes .

• Washing: Wash fixed tissue in 0.2% PBS-Triton X-100 .

• Blocking: Block for 1 hour in a solution containing 1% BSA and 5% normal serum in 0.2% PBS-Triton X-100 .

• Primary antibody: Incubate overnight at 4°C with CRB2 antibody (typically 5 μg/ml or at dilutions of 1:50-1:500) in 1% BSA and 2% normal serum in 0.2% PBS-Triton X-100 .

• Secondary antibody: After washing, incubate for 1 hour at room temperature with fluorescent secondary antibodies (e.g., Alexa Fluor 488 or 555 at 1:500) and nuclear counterstain (e.g., TOPRO-3 at 1:1000) .

• Mounting: Mount samples using an anti-fading reagent .

What is the role of CRB2 in podocyte function and kidney disease?

CRB2 plays a critical role in podocyte function, particularly in mechanotransduction. CRB2 deficiency has been shown to impair podocyte mechanotransduction via disruption of YAP signaling . Knockdown of CRB2 induces YAP activity and target gene expression in podocytes, upregulates YAP-mediated mechanosignaling, and increases the density of focal adhesion and F-actin .

Using Elastic Resonator Interference Stress Microscopy (ERISM), research has demonstrated that CRB2 knockdown enhances podocyte contractility in a substrate stiffness-dependent manner, with the effect decreasing as substrate stiffness increases . This indicates impaired mechanosensing in CRB2-deficient podocytes at low substrate stiffness levels .

Interestingly, CRB2 variants have been associated with autosomal recessive steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (SRNS/FSGS), highlighting its importance in kidney function and disease .

How does CRB2 contribute to retinal development and function?

CRB2 plays a crucial role in the maintenance of retinal neuroepithelium organization, structural integrity, adhesion, photoreceptor polarity, and retinal photoreceptor layer thickness . It is expressed in the retinal pigment epithelium (RPE) and has been identified as a component of the Crumbs complex in the retina .

Studies have shown that CRB2 is specifically localized to cell membranes and the cytoplasm in retinal tissues . The interaction of CRB2 with other proteins in the Crumbs complex, such as PALS1 (protein associated with Lin Seven 1), is essential for proper retinal development and maintenance .

Additionally, CRB2 has been identified as a candidate modifying gene of human CRB1-related retinal dystrophy, suggesting that variations in CRB2 may influence the severity or progression of retinal diseases associated with CRB1 mutations .

How does CRB2 interact with the YAP signaling pathway?

CRB2 functions as an upstream regulator of YAP (Yes-associated protein) activation in the Hippo signaling pathway . In podocytes, CRB2 knockdown induces YAP activity and target gene expression, suggesting that CRB2 normally suppresses YAP signaling . This CRB2-mediated regulation of YAP affects cellular mechanotransduction processes.

The relationship between CRB2 and YAP signaling appears to be substrate stiffness-dependent, with the relative effect of CRB2 knockdown on cell contractility decreasing as substrate stiffness increases . This indicates a complex interplay between CRB2, YAP signaling, and mechanical properties of the cellular environment.

Interestingly, treatment of CRB2 knockdown podocytes with YAP inhibitors (verteporfin and K-975) did not reduce podocyte contractile force development relative to controls, suggesting that additional pathways beyond YAP signaling may contribute to mechanosignaling downstream of CRB2 .

Why might I observe multiple bands for CRB2 in Western blot?

The observation of multiple bands for CRB2 in Western blot is a common phenomenon that can be attributed to several factors:

• Different isoforms: Up to three different isoforms have been reported for CRB2 protein .

• Post-translational modifications: CRB2 undergoes N-glycosylation and potentially other modifications that can alter its migration pattern in SDS-PAGE .

• Tissue-specific expression: In tissue lysates, bands of approximately 150 kDa and above 260 kDa have been detected, while in cell lines, typically only the 150 kDa band is observed .

• Protein complexes or aggregates: The complexity of tissue organization may lead to the formation of protein complexes or aggregates that are not fully dissociated under standard denaturing conditions .

• Proteolytic processing: Partial degradation during sample preparation can result in multiple bands.

To determine which bands represent specific CRB2 detection, validation experiments such as knockdown studies, overexpression studies, and peptide competition assays are recommended .

What controls should I include when using CRB2 antibodies?

When using CRB2 antibodies, the following controls should be included:

• Positive tissue controls: Brain and retina tissues where CRB2 expression has been well-documented .

• Negative controls: Omission of primary or secondary antibodies in immunofluorescence or Western blot experiments .

• Peptide competition assay: Pre-incubation of the CRB2 antibody with the original antigen fusion protein to confirm signal specificity .

• Knockdown controls: Cells transfected with shRNA targeting CRB2 to demonstrate antibody specificity .

• Overexpression controls: Cells overexpressing tagged CRB2 (e.g., CRB2-GFP) to confirm detection of the exogenous protein .

• Cross-reactivity controls: Testing the antibody against related proteins (e.g., CRB3) to ensure specificity within the protein family .

• Loading controls: Use of housekeeping proteins such as β-actin or GAPDH to normalize protein loading .

What are the recommended storage conditions for CRB2 antibodies?

For optimal performance and longevity of CRB2 antibodies, the following storage conditions are recommended:

• Temperature: Store at -20°C .

• Buffer composition: Many commercial CRB2 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Stability: When stored properly, CRB2 antibodies are typically stable for one year after shipment .

• Aliquoting: For small volume antibodies (e.g., 20μL), aliquoting is generally unnecessary for -20°C storage .

• Additives: Some antibody preparations may contain small amounts (0.1%) of BSA as a stabilizer .

Following these storage recommendations will help maintain antibody activity and specificity for experimental applications.

What disease associations have been reported for CRB2?

Several disease associations have been reported for CRB2:

• Kidney diseases: CRB2 variants have been linked to autosomal recessive steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (SRNS/FSGS) .

• Retinal dystrophies: CRB2 has been identified as a candidate modifying gene for human CRB1-related retinal dystrophy, suggesting that variations in CRB2 may influence the severity or progression of retinal diseases .

• Cancer-related processes: CRB2 has been studied in the context of neoplasm metastasis and necrosis, with at least two publications linking CRB2 to these processes .

• Lung diseases: At least one publication has associated CRB2 with lung diseases .

These disease associations highlight the clinical significance of CRB2 and underscore the importance of continued research into its functions and regulation.

What other proteins interact with CRB2 in cellular complexes?

CRB2 interacts with several proteins to form functional complexes in cells:

• PALS1 (MPP5): Protein associated with Lin Seven 1, a key component of the Crumbs complex that interacts with CRB2 in epithelial tissues .

• MPP3: A membrane protein that has been associated with CRB2 in at least one publication .

• MPDZ: Multiple PDZ domain protein that has been studied in relation to CRB2 .

• INADL: InaD-like protein that interacts with components of the Crumbs complex .

• PSEN1: Presenilin-1, which has been associated with CRB2 in published research .

• TP53BP1: Tumor protein p53 binding protein 1, which has been co-mentioned with CRB2 in multiple publications .

• CRB1: Another member of the Crumbs family that is functionally related to CRB2, with over 18 publications discussing both proteins .

These interactions form the basis of CRB2's roles in cell polarity, adhesion, and tissue organization.

What emerging techniques are advancing CRB2 research?

Recent advancements in research techniques have expanded our understanding of CRB2 function:

• Elastic Resonator Interference Stress Microscopy (ERISM): This technique has been used to measure podocyte contractility in the context of CRB2 knockdown, revealing substrate stiffness-dependent effects on cell mechanics .

• Custom-designed antibodies: Development of highly specific antibodies that can distinguish CRB2 from other Crumbs family proteins has improved detection capabilities .

• Gene knockdown approaches: Use of shRNA targeting specific sequences of CRB2 mRNA has provided insights into the functional consequences of CRB2 deficiency .

• Overexpression systems with fusion tags: Expression of CRB2 fused to GFP or other tags has facilitated visualization and tracking of the protein in living cells .

• Pharmacological manipulation: Use of YAP inhibitors such as verteporfin and K-975 in conjunction with CRB2 knockdown has helped elucidate the relationship between CRB2 and YAP signaling pathways .

These techniques continue to enhance our understanding of CRB2's multifaceted roles in cellular function and disease pathogenesis.