BBS10 Antibody

What is BBS10 and what are its key cellular functions?

BBS10 (Bardet-Biedl syndrome 10) is a molecular chaperone belonging to the TCP-1 chaperonin family. It functions primarily to assist protein folding through ATP hydrolysis mechanisms. As a component of the BBS/CCT complex, BBS10 plays a critical role in the assembly of the BBSome, which is essential for ciliogenesis and the regulation of vesicular transport to cilia . Research has demonstrated that BBS10 is also involved in adipogenic differentiation, indicating its broader physiological significance beyond ciliary functions .

Defects in BBS10 are causative for Bardet-Biedl syndrome type 10, a ciliopathy characterized by retinal dystrophy, obesity, polydactyly, renal abnormalities, and cognitive impairment . The protein has a calculated molecular weight of 81 kDa, which corresponds with its observed molecular weight in experimental conditions .

What applications are BBS10 antibodies validated for in research settings?

BBS10 antibodies have been validated for multiple research applications with varying specificity and sensitivity profiles. Based on current validation data, these applications include:

For optimal results, researchers should select antibodies validated specifically for their intended application. Published literature has successfully employed BBS10 antibodies in knockout/knockdown validation studies, further confirming specificity in these experimental contexts .

What are the recommended dilutions and experimental conditions for BBS10 antibody applications?

Optimal dilutions for BBS10 antibodies vary by application and specific antibody preparation. Current recommended dilutions are:

For immunohistochemistry applications, antigen retrieval conditions significantly impact staining outcomes. The recommended protocol involves using TE buffer at pH 9.0, though citrate buffer at pH 6.0 can serve as an alternative . For all applications, antibody titration in each specific experimental system is strongly advised to achieve optimal signal-to-noise ratios and specific binding .

How should BBS10 antibodies be stored and handled to maintain activity?

Proper storage and handling of BBS10 antibodies are crucial for maintaining immunoreactivity and ensuring experimental reproducibility. Most commercially available BBS10 antibodies are provided in a liquid form containing preservatives and stabilizers:

• Storage buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Storage temperature: -20°C (stable for one year after shipment)

• Aliquoting requirements: Not necessary for -20°C storage, though some preparations (20μl sizes) may contain 0.1% BSA

For HRP-conjugated BBS10 antibodies, additional storage considerations include avoiding repeated freeze-thaw cycles. Upon receipt, these antibodies should be stored at either -20°C or -80°C for maximum stability . The presence of stabilizers like glycerol and preservatives such as Proclin 300 (0.03%) help maintain antibody functionality during storage periods .

How can BBS10 antibodies be used to investigate primary cilia structure and function?

BBS10 antibodies are valuable tools for investigating ciliary structures and associated pathologies. Research has demonstrated that BBS10 mutations can affect both cilia length and quantity, making antibody-based detection methods particularly informative for phenotypic characterization .

For cilia visualization and quantification:

• Co-labeling approach: BBS10 antibodies can be used alongside ciliary markers such as acetylated tubulin (AcTub) and ARL13B to identify primary cilia in both undifferentiated cells and during differentiation processes .

• Quantitative analysis parameters:

  • Percentage of ciliated cells (typically counting 200-1,500 cells per condition)

  • Cilia length measurements (using distribution analysis methods like Kolmoglorov-Smirnov tests)

  • Ultrastructural characterization via transmission electron microscopy

In studies of BBS10 mutant cell lines, significant differences in cilia morphology have been detected. For example, in day 7 differentiated cells, certain BBS10 mutations resulted in significantly fewer ciliated cells compared to healthy controls (p = 0.0018) . Immunofluorescence analysis of primary cilia requires careful optimization of fixation protocols to preserve delicate ciliary structures while maintaining antibody epitope accessibility.

What methodological considerations are important when using BBS10 antibodies in kidney organoid models?

Kidney organoid models provide valuable systems for studying BBS10 function in renal development and disease. When using BBS10 antibodies in these complex 3D culture systems, several methodological considerations are critical:

• Expression pattern analysis: qPCR analysis of BBS10 mRNA levels should be performed in parallel with antibody-based protein detection to confirm expression changes during differentiation . Primers should be designed to attach to the 3' end of the transcript and avoid annealing directly over mutation sites .

• Sectioning techniques: For dense kidney organoids at day 20 of differentiation, 18μm cryosections are recommended for optimal antibody penetration and imaging resolution .

• Marker combinations: Co-staining with nephron segmentation markers is essential to contextualize BBS10 expression patterns within developing renal structures. This approach has revealed that certain BBS10 mutations can lead to abnormal organoid morphology with irregular structures, flattened regions, and lack of rounded, domed architecture .

• Controls and validation:

  • Use of multiple healthy donor lines (pooled data from lines such as Cuhk_1, Hoik_1, Kegd_2) for robust comparison

  • Multiple independent experiments (n=3-5) for each condition to account for batch effects

Research has demonstrated that while BBS10 mutations might not significantly impact the capacity of cells to differentiate into WT1-positive kidney progenitors, they can affect ciliary features critical for proper nephron formation and function .

What strategies can be employed to validate BBS10 antibody specificity?

Validating antibody specificity is critical for ensuring reliable experimental results, particularly when studying BBS10 in disease models. Several complementary approaches are recommended:

• Genetic validation strategies:

  • CRISPR/Cas9-mediated knockout: Using pSpCas9(BB)-2A-GFP (PX458) plasmid systems targeting different locations within the BBS10 gene, particularly sites in the first exon where deletions or frameshifts would significantly impact protein expression .

  • RNA interference: siRNA or shRNA knockdown followed by Western blot analysis to confirm reduced protein levels .

• Recombinant protein controls:

  • Using antibodies raised against defined recombinant fragments (e.g., BBS10 fusion protein Ag3073) for validation experiments .

  • Immunoprecipitation followed by mass spectrometry to confirm target specificity.

• Cross-validation with multiple antibodies:

  • Comparing monoclonal (e.g., Mouse IgG2b, 66679-1-Ig) and polyclonal (e.g., Rabbit IgG, 12421-2-AP) antibodies targeting different epitopes of BBS10 .

  • Confirmation with independent antibody clones showing consistent patterns.

The literature demonstrates successful validation through published applications in knockout/knockdown studies . When designing validation experiments, researchers should consider the specific immunogen used to generate the antibody, as this influences epitope recognition and potential cross-reactivity.

How do BBS10 mutations affect antibody binding and experimental design?

BBS10 mutations associated with Bardet-Biedl syndrome can significantly impact antibody binding and experimental outcomes, requiring careful consideration in study design:

• Epitope accessibility issues:

  • Mutations may alter protein folding, affecting epitope exposure and antibody recognition

  • For mutations near the N-terminus, antibodies targeting C-terminal epitopes may be more reliable for detecting mutant proteins

• Expression level variations:

  • qPCR analysis has shown that BBS10 mRNA levels can vary significantly between healthy and mutant lines during differentiation processes

  • Different BBS10 mutations (e.g., in lines Laig, Xiry, and Nolz_4) show distinct expression patterns requiring individualized experimental approaches

• Functional readouts:

  • Beyond simple detection, assessing ciliary phenotypes (length, number) provides functional validation

  • Comparison of cellular phenotypes across multiple BBS10 mutant lines reveals genotype-specific effects

For accurate interpretation of results, using multiple detection methods is recommended, including Western blotting for protein size and abundance, immunofluorescence for localization patterns, and functional assays specific to ciliary dynamics .

What are the best practices for using BBS10 antibodies in multicolor immunofluorescence studies?

Multicolor immunofluorescence offers powerful insights into BBS10 localization and interactions with other cellular components. Optimization of these complex protocols requires attention to several technical considerations:

• Antibody compatibility:

  • Mouse monoclonal BBS10 antibodies (e.g., 66679-1-Ig) pair effectively with rabbit polyclonal antibodies against other ciliary markers

  • Rabbit polyclonal BBS10 antibodies (e.g., 12421-2-AP) work well with mouse monoclonal antibodies against structural proteins

• Validated co-staining combinations:

  • BBS10 + Acetylated Tubulin (AcTub) + ARL13B for ciliary studies

  • BBS10 + WT1 for kidney progenitor identification

  • Cell Mask + DAPI for visualizing cell shape and nuclear morphology in conjunction with BBS10

• Imaging parameters:

  • High-content imaging platforms with image analysis software enable measurement of multiple cellular features simultaneously

  • For quantitative analysis, collecting data on 14+ features per cell across multiple fields is recommended

• Statistical analysis approaches:

  • Principal component analysis (PCA) for identifying patterns in multivariate data

  • Multivariate logistic regression for genotype-phenotype correlations

  • Kolmoglorov-Smirnov tests for comparing distributions between healthy and BBS10 mutant samples

Studies have successfully employed these approaches to detect subtle phenotypic differences in BBS10 mutant cells that might be overlooked with single-marker analysis, particularly in the context of ciliary length and differentiation capacity .

What are common pitfalls when working with BBS10 antibodies and how can they be addressed?

Researchers frequently encounter challenges when working with BBS10 antibodies. Common pitfalls and their solutions include:

• Nonspecific binding in Western blot applications:

  • Optimize antibody dilution (test range from 1:500 to 1:6000)

  • Extend blocking time and increase blocking agent concentration

  • Verify expected molecular weight (81 kDa) against appropriate markers

  • Include positive control samples (HepG2, A549, MCF-7, NCI-H1299 cells)

• Weak immunohistochemistry signals:

  • Test both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Extend primary antibody incubation time at 4°C

  • Utilize signal amplification systems for low-abundance detection

• Background in immunofluorescence applications:

  • Increase washing steps with PBS containing 0.1% Tween-20

  • Optimize fixation conditions (test both paraformaldehyde and methanol fixation)

  • Pre-adsorb antibodies with cell/tissue homogenates from irrelevant species

• Batch-to-batch variation:

  • Validate each new antibody lot against previously successful lots

  • Maintain consistent experimental conditions across experiments

  • Document antibody information including catalog number and RRID (e.g., AB_2882033, AB_2064962)

Addressing these challenges requires systematic optimization and careful experimental design. Maintaining detailed records of successful protocols facilitates reproducibility across experiments and research groups.

How can researchers quantitatively assess BBS10 expression levels in different experimental systems?

Quantitative assessment of BBS10 expression requires complementary approaches for comprehensive analysis:

• Western blot quantification:

  • Utilize housekeeping proteins (GAPDH, β-actin) for normalization

  • Employ digital imaging systems with linear dynamic range

  • Perform densitometric analysis with appropriate controls

  • Test multiple antibody dilutions (1:500-1:6000) to ensure linear response range

• Quantitative PCR approaches:

  • Normalize to stable reference genes (GAPDH, 18S) tested across experimental conditions

  • Design primers to avoid mutation sites in BBS10 variants

  • Perform technical triplicates and biological replicates (n=3-5 independent experiments)

  • Use relative quantification methods with appropriate statistical analysis

• High-content imaging methods:

  • Measure multiple parameters per cell (≥14 features recommended)

  • Calculate various statistical measures per well (mean, standard deviation, sum, maximum, minimum, median values)

  • Implement Principal Component Analysis for complex datasets

  • Employ appropriate statistical tests for distribution comparisons (e.g., Kolmoglorov-Smirnov test)

For comprehensive analysis, researchers should combine protein-level detection (antibody-based) with transcript-level quantification, particularly when studying BBS10 mutations that might affect protein stability but not mRNA levels .

How are BBS10 antibodies being utilized in ciliopathy research beyond Bardet-Biedl syndrome?

BBS10 antibodies are increasingly applied to broader ciliopathy research, yielding insights into fundamental ciliary biology and disease mechanisms:

• Cilia structure-function relationships:

  • Investigation of how BBS10 mutations affect basal body organization and axoneme structure

  • Analysis of dynein arm integrity and microtubule central pair formation in BBS10 mutant cilia

  • Correlation of ultrastructural defects with functional ciliary transport abnormalities

• Cellular differentiation studies:

  • Monitoring BBS10 expression during cell differentiation processes

  • Investigating BBS10's role in adipogenic differentiation pathways

  • Assessing how ciliary defects impact cellular fate decisions in development

• Organoid research applications:

  • Using kidney organoids to model renal defects associated with ciliopathies

  • Comparing BBS10-dependent phenotypes across different organ-specific organoid systems

  • Developing standard protocols for antibody-based assessment of ciliary function in 3D culture systems

These research directions leverage BBS10 antibodies not only as diagnostic tools but as mechanistic probes for understanding fundamental biological processes dependent on primary cilia function. The combination of traditional antibody applications with advanced imaging techniques and genetic models is advancing our understanding of ciliopathies beyond classical clinical manifestations.

What role do BBS10 antibodies play in investigating potential therapeutic approaches for ciliopathies?

BBS10 antibodies serve as critical tools in the development and assessment of therapeutic strategies for ciliopathies:

• Target validation studies:

  • Confirming BBS10 expression levels before and after therapeutic interventions

  • Tracking BBS10 protein localization changes in response to treatments

  • Identifying potential off-target effects of therapies on BBSome assembly and function

• Gene therapy monitoring:

  • Assessing BBS10 protein restoration following gene delivery approaches

  • Verifying proper subcellular localization of transgene-derived BBS10 protein

  • Quantifying functional recovery of ciliary structures using immunofluorescence-based assays

• Small molecule screening:

  • Evaluating compound effects on BBS10 protein levels and stability

  • Monitoring changes in BBS10 interactions with other BBSome components

  • Assessing recovery of ciliary morphology and function using antibody-based readouts

• Mechanistic studies:

  • Investigating how chaperonin function modulation might compensate for BBS10 defects

  • Exploring BBSome assembly pathways as potential intervention points

  • Examining cross-talk between BBS10-mediated processes and other cellular pathways

By providing specific and sensitive detection methods, BBS10 antibodies enable researchers to evaluate therapeutic efficacy at molecular, cellular, and tissue levels, accelerating the development of interventions for patients with Bardet-Biedl syndrome and related ciliopathies.