BBS9 Antibody

What is the BBS9 protein and what is its functional significance in cellular processes?

BBS9 (also known as PTHB1, Parathyroid hormone-responsive B1 gene protein) is a critical component of the BBSome complex, which functions as a coat complex required for trafficking specific membrane proteins to the primary cilia . The protein plays essential roles in:

• Ciliogenesis (though it is dispensable for centriolar satellite function)

• Promoting proper BBSome complex assembly and ciliary localization

• Mediating interaction with Rab8 GDP/GTP exchange factor (RAB3IP/Rabin8) at the basal body

• Supporting ciliary membrane extension through Rab8(GTP)-mediated docking and fusion of carrier vesicles

BBS9 is particularly significant in disease contexts as mutations in the BBS9 gene are associated with Bardet-Biedl Syndrome, a genetically heterogeneous disorder characterized by retinopathy, obesity, cognitive impairment, and renal abnormalities . Individuals with biallelic truncation variants in BBS9 typically display primary manifestations of Bardet-Biedl Syndrome, particularly retinal dystrophy .

What are the validated applications for BBS9 antibodies in research?

BBS9 antibodies have been validated for multiple research applications as detailed in the following table:

It is strongly recommended that researchers titrate antibodies in their specific experimental systems to achieve optimal results, as performance can be sample-dependent .

How should researchers select the appropriate BBS9 antibody for their specific experimental needs?

Selection criteria should include:

• Target epitope location: Consider whether your experiment requires detection of a specific domain or region of BBS9. For example, Abcam's antibody (ab234818) targets amino acids 1-200 , while Sigma's antibody (HPA021289) targets a different immunogen sequence .

• Validated applications: Ensure the antibody has been validated for your intended application. While some antibodies work across multiple applications, others may be optimized for specific techniques.

• Species reactivity: Confirm that the antibody recognizes BBS9 in your experimental model. Currently available antibodies show validated reactivity with human and mouse samples .

• Antibody format: Consider whether a polyclonal (offering broader epitope recognition) or monoclonal (higher specificity) antibody better suits your experimental design. Most currently available BBS9 antibodies are rabbit polyclonals .

• Published validation: Review literature utilizing the antibody in similar experimental contexts. Several publications have successfully employed BBS9 antibodies in Western blotting and immunofluorescence applications .

What controls should be included when working with BBS9 antibodies?

Proper experimental controls are essential for ensuring valid and interpretable results:

• Positive controls: Include samples with known BBS9 expression. Validated positive samples include HEK-293 cells, mouse testis tissue, human heart tissue, HeLa cells, Jurkat cells, and mouse heart tissue for Western blotting applications .

• Negative controls: Consider using BBS9 knockdown/knockout samples where available, or tissues known to have minimal BBS9 expression.

• Loading and procedural controls: Include standard loading controls for Western blots (β-actin, GAPDH) and procedural controls for immunostaining (secondary antibody-only controls).

• Blocking peptide controls: Where available, use the immunizing peptide to confirm antibody specificity.

• Subcellular localization validation: For immunofluorescence experiments, co-staining with established ciliary markers (such as acetylated tubulin) can help confirm the expected localization pattern of BBS9 at the base of primary cilia.

How can researchers optimize detection of BBS9 in the context of BBSome complex studies?

The BBSome complex plays a crucial role in ciliary trafficking, and optimizing BBS9 detection in this context requires:

• Co-immunoprecipitation optimization: When studying BBSome assembly, use mild lysis conditions to preserve protein-protein interactions. PBS-based buffers with 0.1-0.5% NP-40 or Triton X-100 are often effective. BBS9 antibodies can be used to pull down the entire BBSome complex, allowing analysis of associated components.

• Proximity ligation assays: Consider using this technique to detect and visualize interactions between BBS9 and other BBSome components (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBIP10) or interacting partners like RAB3IP/Rabin8.

• Subcellular fractionation: To enrich for ciliary or centrosomal fractions before Western blotting or immunoprecipitation, as BBS9 is specifically localized to these structures.

• Dual immunofluorescence: Co-stain with antibodies against other BBSome components and ciliary markers to confirm the expected co-localization patterns and assess BBSome integrity in various experimental conditions.

• Functional trafficking assays: Use BBS9 antibodies alongside markers of ciliary membrane proteins to assess the impact of experimental manipulations on the BBSome's trafficking function .

What approaches can be used to address the discrepancy between calculated and observed molecular weights of BBS9?

The observed molecular weight of BBS9 in Western blots (~60 kDa) differs significantly from its calculated weight (99 kDa) . Researchers should consider:

• Alternative splicing: BBS9 has multiple transcript variants. Verify which isoform is predominant in your experimental system and whether your antibody's epitope is present in all isoforms.

• Post-translational modifications: Investigate whether proteolytic processing affects the observed size. Consider using phosphatase treatment or other enzymatic approaches to assess the impact of post-translational modifications.

• Sample preparation optimization:

  • Test different lysis buffers to ensure complete extraction

  • Vary denaturation conditions (temperature, time, reducing agents)

  • Include protease inhibitors to prevent degradation

  • Consider native vs. denaturing conditions to assess complex formation

• Gradient gels: Utilize gradient gels for better resolution of the protein of interest.

• Validation with recombinant protein: Run purified or recombinant BBS9 alongside your samples as a size reference.

How can BBS9 antibodies be leveraged to investigate ciliopathy disease mechanisms?

BBS9 antibodies are valuable tools for investigating Bardet-Biedl Syndrome and related ciliopathies:

• Patient-derived samples: Use BBS9 antibodies to assess protein expression and localization in cells derived from individuals with BBS9 mutations. Compare with wild-type controls to characterize pathogenic mechanisms.

• Genotype-phenotype correlations: For known BBS9 variants, correlate protein expression levels or localization patterns with clinical severity to identify functional domains critical for disease pathogenesis.

• Rescue experiments: In cellular models with BBS9 mutations, assess whether wild-type BBS9 expression restores normal BBSome assembly and trafficking using immunofluorescence and co-immunoprecipitation with BBS9 antibodies.

• Tissue-specific effects: Examine BBS9 expression and localization in different tissues (retina, kidney, brain) using immunohistochemistry to better understand tissue-specific manifestations of disease.

• Interaction with disease modifiers: Use co-immunoprecipitation with BBS9 antibodies to identify genetic modifiers that interact with BBS9 and potentially influence disease severity.

Research has shown that biallelic truncation variants in BBS9 are associated with primary signs of Bardet-Biedl Syndrome, particularly retinal dystrophy . Immunohistochemical analysis can help determine whether specific mutations affect protein stability, localization, or interaction capabilities.

What methodological considerations are important when using BBS9 antibodies in immunofluorescence studies of primary cilia?

Primary cilia are delicate structures requiring specific considerations for optimal imaging:

• Fixation protocol optimization:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves most ciliary structures

  • Avoid methanol fixation which can disrupt membrane structures

  • For some applications, glutaraldehyde (0.1-0.5%) may better preserve ciliary ultrastructure

• Permeabilization considerations:

  • Gentle permeabilization (0.1-0.2% Triton X-100 or 0.1% saponin) to maintain ciliary integrity

  • Brief permeabilization times (5-10 minutes) to prevent over-extraction

• Blocking optimization:

  • Extended blocking (1-2 hours) with 5-10% normal serum from the same species as the secondary antibody

  • Addition of 0.1-0.3% BSA can reduce non-specific binding

• Co-staining recommendations:

  • Acetylated α-tubulin or ARL13B as ciliary shaft markers

  • γ-tubulin or pericentrin as basal body markers

  • Include these markers to confirm the ciliary localization of BBS9

• Image acquisition settings:

  • High-magnification confocal microscopy (63x or 100x objectives)

  • Z-stack acquisition to capture the entire ciliary structure

  • Deconvolution processing to enhance resolution of ciliary details

• Quantification approaches:

  • Measure ciliary length, frequency, and BBS9 signal intensity

  • Assess co-localization with other BBSome components

  • Compare wild-type vs. disease models for alterations in BBS9 distribution

The recommended dilution range for immunofluorescence applications is 1:10-1:100, though this should be optimized for specific experimental conditions .

What are common challenges in Western blot detection of BBS9 and how can they be addressed?

Researchers frequently encounter several challenges when detecting BBS9 by Western blot:

• Inconsistent band size: As noted, BBS9's observed molecular weight (~60 kDa) differs from its calculated size (99 kDa) . To address this:

  • Run a gradient gel (4-20%) to better resolve potential isoforms

  • Include positive control samples with known BBS9 expression

  • Consider testing multiple BBS9 antibodies targeting different epitopes

• Weak signal intensity:

  • Increase antibody concentration (within recommended range: 1:500-1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Increase protein loading (50-100 μg total protein)

• High background:

  • Extend washing steps (4-5 washes, 10 minutes each)

  • Increase blocking time and concentration (5% non-fat dry milk or BSA)

  • Dilute primary antibody in fresh blocking buffer

  • Consider using more stringent washing conditions (higher salt concentration)

• Multiple non-specific bands:

  • Use fresher antibody aliquots to avoid degradation

  • Include additional protease inhibitors in sample preparation

  • Optimize antibody concentration and incubation time

  • Consider using gradient gels for better resolution

• Sample preparation considerations:

  • Include phosphatase inhibitors if phosphorylation affects detection

  • Use fresh samples when possible

  • Avoid repeated freeze-thaw cycles of protein lysates

How can immunohistochemical detection of BBS9 be optimized in tissue sections?

For optimal immunohistochemical detection of BBS9 in tissue sections:

• Antigen retrieval optimization:

  • Recommended: TE buffer pH 9.0 (primary option)

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval (pressure cooker or microwave) is often more effective than enzymatic methods

• Antibody concentration:

  • Recommended dilution range: 1:20-1:200 or 1:20-1:50

  • Titrate for each tissue type and fixation method

  • Perform a dilution series on control tissues to determine optimal concentration

• Incubation conditions:

  • Extended primary antibody incubation (overnight at 4°C) often yields better results than shorter incubations

  • Humid chamber to prevent section drying

• Signal amplification:

  • Consider biotin-streptavidin systems or polymer-based detection for low-abundance targets

  • Tyramide signal amplification for particularly challenging samples

• Counterstaining considerations:

  • Light hematoxylin counterstaining to avoid obscuring specific signal

  • Clear differentiation to reduce background

• Controls to include:

  • Positive control tissue (human liver has been validated)

  • Negative controls (primary antibody omission, isotype controls)

  • Absorption controls with immunizing peptide where available

What strategies can improve success rates in co-immunoprecipitation experiments using BBS9 antibodies?

Co-immunoprecipitation (Co-IP) of BBS9 and its interacting partners requires careful optimization:

• Antibody selection and amount:

  • Recommended: 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Consider crosslinking the antibody to beads to eliminate heavy/light chain interference in detection

• Lysis buffer optimization:

  • Use mild, non-denaturing buffers to preserve protein-protein interactions

  • Recommended: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 or 0.5% Triton X-100

  • Include protease and phosphatase inhibitors

  • Consider adding specific proteasome inhibitors (MG132) if protein stability is an issue

• Incubation conditions:

  • Extended incubation (4-16 hours at 4°C) with gentle rotation

  • Optimize antibody-lysate binding time vs. non-specific interactions

• Washing stringency balance:

  • Sufficient washing to remove non-specific binding

  • Not so stringent as to disrupt legitimate interactions

  • Consider detergent concentration gradients in wash buffers

• Detection strategies:

  • Immunoblot for both BBS9 and suspected interacting partners

  • Use clean detection systems (TrueBlot® secondary antibodies) to avoid heavy/light chain interference

  • Consider mass spectrometry for unbiased identification of co-precipitated proteins

• Reciprocal Co-IP validation:

  • Confirm interactions by performing reciprocal Co-IP (using antibodies against interacting partners)

  • This approach is particularly important for validating novel interactions with other BBSome components

Mouse testis tissue has been validated for successful immunoprecipitation of BBS9 and may serve as a positive control for method optimization.

How can BBS9 antibodies be used to investigate the pathophysiology of Bardet-Biedl Syndrome?

BBS9 antibodies provide valuable tools for investigating Bardet-Biedl Syndrome mechanisms:

• Expression analysis in patient samples:

  • Compare BBS9 protein levels between patient-derived cells and controls

  • Assess localization patterns in different cellular compartments

  • Determine whether specific mutations affect protein stability or subcellular distribution

• Functional studies in disease models:

  • Examine BBSome complex integrity in BBS9 mutant backgrounds

  • Assess ciliary trafficking defects through co-localization studies

  • Investigate downstream signaling pathway disruptions

• Genotype-phenotype correlations:

  • Compare protein expression and localization patterns across different BBS9 variants

  • Correlate molecular findings with clinical manifestations

  • Recent research has identified biallelic truncation variants in BBS9 associated with retinal dystrophy, a primary sign of BBS

• Therapeutic development:

  • Use BBS9 antibodies to assess the efficacy of experimental therapies in restoring normal protein expression or localization

  • Monitor BBSome complex assembly and function in response to treatments

• Molecular diagnostics:

  • Develop immunohistochemical or immunofluorescence approaches to identify BBS9-related ciliary defects in accessible patient samples

  • Establish correlations between immunostaining patterns and genetic findings

What considerations are important when analyzing BBS9 expression in different tissue contexts?

BBS9 expression analysis across tissues requires specific methodological considerations:

• Tissue-specific optimization:

  • Adjust fixation protocols based on tissue type (epithelial vs. neural vs. connective tissues)

  • Optimize antigen retrieval methods for each tissue context

  • Validated human tissues include liver for IHC and heart for Western blot

• Expression pattern interpretation:

  • BBS9 typically localizes to the base of primary cilia and centrosomes

  • Altered localization patterns may indicate pathological processes

  • Quantify both expression level and subcellular distribution

• Co-expression analysis:

  • Pair BBS9 detection with tissue-specific markers

  • In retinal tissue, combine with photoreceptor markers

  • In kidney sections, combine with renal tubule markers

• Developmental considerations:

  • BBS9 expression and localization may vary during development

  • Temporal analysis may reveal critical periods for BBS9 function

  • Compare findings across developmental stages when possible

• Species-specific differences:

  • Currently validated reactivity includes human and mouse samples

  • Consider potential differences in expression patterns across species

  • Use appropriate positive controls for each species

• Technical validation:

  • Confirm antibody specificity in each tissue context

  • Include appropriate controls (positive, negative, absorption)

  • Consider multiple detection methods to corroborate findings

How can BBS9 antibodies contribute to understanding non-canonical functions of the BBSome complex?

Beyond its established role in ciliary trafficking, emerging research suggests additional functions for the BBSome complex and its components:

• Non-ciliary functions:

  • Use BBS9 antibodies to investigate potential roles in non-ciliated cells

  • Examine subcellular localization in various cellular compartments

  • Study potential functions in vesicular trafficking outside the ciliary context

• Signaling pathway interactions:

  • Investigate BBS9's role in Wnt, Hedgehog, and other signaling pathways

  • Use proximity ligation assays with BBS9 antibodies to identify novel interacting partners

  • Analyze co-localization with signaling pathway components in different cellular contexts

• Cell cycle regulation:

  • Examine BBS9 expression and localization throughout the cell cycle

  • Investigate potential functions in centrosome regulation beyond ciliary roles

  • Compare proliferating vs. quiescent cells for differences in BBS9 distribution

• Tissue-specific functions:

  • Use immunohistochemistry to map BBS9 expression across diverse tissues

  • Correlate expression patterns with tissue-specific phenotypes in BBS

  • Identify potential tissue-specific interacting partners

• Developmental roles:

  • Analyze BBS9 expression during embryonic and post-natal development

  • Investigate potential roles in developmental signaling cascades

  • Correlate developmental expression patterns with congenital manifestations of BBS

What technologies can enhance BBS9 detection in challenging experimental contexts?

Advanced techniques to improve BBS9 detection in difficult contexts:

• Super-resolution microscopy:

  • STED, STORM, or PALM approaches for nanoscale localization of BBS9

  • Improved resolution of BBSome complex arrangement at the ciliary base

  • Multi-color super-resolution to visualize protein-protein interactions

• Proximity labeling approaches:

  • BioID or APEX2 fusions with BBS9 to identify proximal proteins

  • Temporal mapping of BBS9 interaction networks

  • Identification of transient or weak interactions missed by co-immunoprecipitation

• Live-cell imaging:

  • Correlation of fixed-cell antibody staining with live-cell fluorescent protein fusions

  • Validation of dynamic behaviors observed in live imaging with antibody-based approaches

  • FRAP (Fluorescence Recovery After Photobleaching) studies paired with immunofluorescence

• Mass spectrometry enhancement:

  • Use BBS9 antibodies for immunoprecipitation followed by mass spectrometry

  • Targeted proteomics approaches to detect specific BBS9 peptides

  • Post-translational modification mapping to better understand regulatory mechanisms

• Organ-on-chip and 3D culture systems:

  • Immunofluorescence optimization for 3D cultures and organoids

  • Development of clearing protocols compatible with BBS9 immunostaining

  • Correlation of in vitro findings with in vivo tissues

These emerging approaches, combined with established antibody-based techniques, provide powerful tools for advancing our understanding of BBS9 function in health and disease.