Sdccag8 Antibody

What is SDCCAG8 and why is it significant in research?

SDCCAG8, also known as Centrosomal Colon Cancer Autoantigen Protein (CCCAP), is a 713 amino acid cytoplasmic protein that exists as a homodimer and localizes primarily to centrioles. It is expressed in multiple tissues including thymus, prostate, testis, ovary, small intestine, colon, mucosa, and renal cancer tumors . The protein is encoded by a gene located on human chromosome 1, which spans approximately 260 million base pairs and contains over 3,000 genes .

SDCCAG8 has gained significant research interest because of its association with nephronophthisis-related ciliopathies (NPHP-RC), a recessive disorder characterized by dysplasia or degeneration of the kidney, retina, and cerebellum . The protein interacts with oral-facial-digital syndrome 1 (OFD1) and has been implicated in ciliary function, making it a crucial target for understanding ciliopathies and their associated pathologies .

What are the most commonly used SDCCAG8 antibodies and their specific applications?

Researchers typically use two main types of SDCCAG8 antibodies:

• Monoclonal antibodies: These offer high specificity for SDCCAG8 detection. The mouse monoclonal antibody derived from immunization with SDCCAG8 recombinant protein has been validated for Western blot applications in various cell lines including HEK-293, COLO 320, and THP-1 cells, with an observed molecular weight of approximately 83 kDa .

• Polyclonal antibodies: These detect endogenous levels of total SDCCAG8 protein and are often used for broader detection capabilities across multiple experimental conditions .

Applications validated for SDCCAG8 antibodies include:

• Western blotting (1:500-1:5000 dilution)

• ELISA

• Immunofluorescence for centrosomal localization studies

• Co-immunoprecipitation assays for protein interaction studies

How should samples be prepared for optimal SDCCAG8 detection?

For optimal detection of SDCCAG8 in immunoblotting experiments:

• Cell lysate preparation:

  • Use IP lysis buffer for cell extraction

  • Centrifuge samples at 16,000 × g for 30 minutes at 4°C to obtain cleared lysates

• SDS-PAGE conditions:

  • 4-12% Bis-Tris gels in MOPS buffer provide optimal resolution for the 83 kDa SDCCAG8 protein

  • Transfer to nitrocellulose membrane using standard protocols

• Immunoblotting conditions:

  • Block membranes in TBS containing 5% milk and 0.1% Tween-20

  • Use recommended antibody dilutions (1:500-1:5000 for Western blot)

  • Store antibodies at -20°C and DO NOT ALIQUOT to maintain stability

For immunofluorescence studies:

• Fixation methods should preserve centrosomal structures

• Co-staining with centrosomal markers such as γ-tubulin, ninein, or CEP164 helps confirm specific localization

Where does SDCCAG8 localize within the cell and how can this be visualized?

SDCCAG8 demonstrates a specific centrosomal localization pattern with distinct characteristics:

• Centrosomal positioning: SDCCAG8 localizes to centrosomes but in a position set apart from the γ-tubulin signal that marks centrioles and from the CEP164 signal that marks distal centrosomal appendages .

• Colocalization patterns:

  • Tight colocalization with ninein, a marker of centrosomal appendages

  • Colocalization with NPHP5/IQCB1 and OFD1, proteins also implicated in NPHP-RC

  • In photoreceptor cells, SDCCAG8 is located in the transition zone, distal to the basal body marker γ-tubulin and distal to but clearly separated from the pericentriolar marker CEP290

• Visualization techniques:

  • Immunofluorescence using specific anti-SDCCAG8 antibodies

  • Co-staining with established centrosomal/basal body markers

  • High-resolution microscopy to differentiate the distinct localization patterns

The subcellular localization of SDCCAG8 provides important insights into its functional role in centrosome biology and ciliogenesis.

What protein interactions have been identified for SDCCAG8 and how can these be studied?

SDCCAG8 has been shown to interact with several proteins through various experimental approaches:

• Confirmed interaction partners:

  • RABEP2: A RAB effector protein

  • ERC1: Another RAB effector protein

  • CEP131: A centrosomal satellite protein

  • OFD1: A centrosomal protein mutated in oral-facial-digital syndrome

• Methods for studying SDCCAG8 interactions:

  • Co-immunoprecipitation (Co-IP): Successfully used to demonstrate interaction between SDCCAG8 and its partners

  • GST pull-down assays: Validated the interaction between SDCCAG8 and OFD1

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture): Identified interaction partners grouped into functional categories including centriolar satellite components, endosomal vesicle components, tRNA synthesis complex proteins, and myosin type II motors involved in ciliogenesis

• Domain-specific interactions:

  • The C-terminal region of SDCCAG8 (aa 533-713) containing two predicted coiled-coil motifs interacts with the C-terminal region of OFD1 (aa 615-1012) containing the last two of six predicted coiled-coil motifs

  • Full-length SDCCAG8 is typically required for interaction with partners like RABEP2, ERC1, and CEP131, although the C-terminal fragment shows weak binding to RABEP2

What are the key considerations for SDCCAG8 co-immunoprecipitation experiments?

For successful co-immunoprecipitation (Co-IP) of SDCCAG8 and its interaction partners:

• Experimental setup:

  • Transfect cells with a tagged SDCCAG8 construct (e.g., FLAG-tagged) or use antibodies against endogenous SDCCAG8

  • Include appropriate controls: negative control (IgG), positive control (known interactor), and knockdown control to confirm specificity

• Protocol optimization:

  • Cell lysis: Use IP lysis buffer (Pierce) to preserve protein complexes

  • Centrifugation: 16,000 × g for 30 min at 4°C produces cleared lysates

  • Immunoprecipitation: Anti-FLAG M2 beads (for tagged constructs) or specific antibodies against endogenous proteins

  • SDS-PAGE: 4-12% Bis-Tris gels in MOPS buffer for optimal resolution

• Validation controls:

  • Reciprocal Co-IP: As demonstrated in the FLAG-SDCCAG8 and CEP131 interaction study, where antibodies against CEP131 successfully co-immunoprecipitated FLAG-SDCCAG8 from control lysates, but not from siCEP131 knockdown lysates

  • Input controls: Always include Western blot analysis of whole cell lysates to confirm equal loading and expression of target proteins

How can domain-specific functions of SDCCAG8 be investigated?

To study the domain-specific functions of SDCCAG8:

• Truncation constructs:

  • Design constructs expressing different domains of SDCCAG8 (N-terminal, C-terminal, and middle portions)

  • The full-length SDCCAG8 contains:

    • An N-terminal globular domain

    • A nuclear localization signal

    • Eight putative coiled-coil domains

• Functional assays:

  • Protein interaction studies using different truncation constructs to map interaction domains

  • Subcellular localization studies to determine which domains are necessary for centrosomal targeting

  • Rescue experiments in SDCCAG8-deficient cells to identify functionally essential domains

• Experimental findings:

  • Full-length SDCCAG8 isoform-a (713 amino acids) localizes to the vicinity of centrosomes

  • Truncated SDCCAG8 constructs generally fail to immunoprecipitate interaction partners like RABEP2, ERC1, and CEP131, except for the C-terminal fragment that shows weak binding to RABEP2

  • The C-terminal SDCCAG8 region (aa 533-713) containing two predicted coiled-coil motifs interacts with the C-terminal region of OFD1

How can SDCCAG8 function be studied in the context of ciliogenesis?

Studying SDCCAG8's role in ciliogenesis requires specialized approaches:

• Knockdown/knockout models:

  • siRNA knockdown of SDCCAG8 in hTERT-RPE1 cells has demonstrated a significant reduction in cilia formation (43% of SDCCAG8 knockdown cells grew cilia compared to 94% of wild-type cells, p=0.0092)

  • Cilia length is significantly reduced in SDCCAG8 knockdown cells (1.9±0.1μ, n=63) compared to wild-type cells (2.8±0.1μ, n=80, p<0.0001)

• Hedgehog signaling assessment:

  • Mouse embryonic fibroblasts (MEFs) from Sdccag8^gt/gt mice show attenuated response to Hedgehog signal agonist SAG, with reduced levels of Hh pathway target gene Gli1

  • This suggests that SDCCAG8 is involved in Hedgehog signaling regulation, a critical pathway in development and ciliogenesis

• Experimental readouts:

  • Cilia formation rate (percentage of ciliated cells)

  • Cilia length measurements

  • Hedgehog pathway target gene expression (e.g., Gli1)

  • Immunofluorescence analysis of cilia structure and ciliary protein localization

What approaches can be used to study SDCCAG8 isoforms and their differential functions?

SDCCAG8 exists in multiple isoforms with potentially distinct functions:

• Isoform characterization:

  • Full-length isoform-a: 3,267 nt, encodes an 82.7-kDa protein (713 amino acids)

  • Additional isoforms include isoforms-b and -e, which can be detected by RT-PCR in hTERT-RPE cells

• Isoform-specific detection:

  • Use antibodies that recognize specific regions of SDCCAG8

  • Antibodies against the C-terminal region can detect both full-length isoform-a and C-terminal short isoform-e

  • RT-PCR with isoform-specific primers can confirm expression at the mRNA level

• Functional differentiation:

  • The full-length isoform-a appears to be the relevant isoform for retinal-renal phenotypes associated with SDCCAG8 mutations

  • Evidence suggests different localization patterns among isoforms, with full-length SDCCAG8 isoform-a localizing to centrosomes while other isoforms may have distinct subcellular distributions

How can conflicting data about SDCCAG8 localization be resolved?

Resolving contradictory findings regarding SDCCAG8 localization requires:

• High-resolution microscopy techniques:

  • Super-resolution microscopy to precisely map SDCCAG8 localization relative to other centrosomal/ciliary markers

  • 3D reconstruction to visualize the spatial relationship between SDCCAG8 and other structures

• Multiple marker analysis:

  • Use a panel of markers for different centrosomal and ciliary compartments

  • Evidence shows SDCCAG8 localizes near but distinct from γ-tubulin (centrioles) and CEP164 (distal appendages), while colocalizing with ninein (centrosomal appendages)

  • In photoreceptors, SDCCAG8 is found in the transition zone, distal to basal body marker γ-tubulin and separate from pericentriolar marker CEP290

• Cell type and context considerations:

  • SDCCAG8 localization patterns may differ between cell types

  • In MDCK-II renal epithelial cells, SDCCAG8 localizes just distal to the centrosomal marker γ-tubulin

  • Consider cell cycle stage, differentiation state, and ciliogenesis stage when interpreting localization data

What are common technical challenges with SDCCAG8 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with SDCCAG8 antibodies:

• Detection sensitivity:

  • SDCCAG8 is expressed at relatively low levels in many tissues, as evidenced by Northern blot analysis showing low expression in mouse liver, spleen, kidney, brain, heart, and muscle

  • Solution: Use sensitive detection methods, optimize antibody concentrations, and consider signal amplification techniques

• Isoform specificity:

  • Multiple SDCCAG8 isoforms exist, and antibodies may recognize different subsets

  • Solution: Characterize antibody specificity using isoform-specific controls and Western blotting to confirm detection of expected isoforms

• Storage and handling:

  • SDCCAG8 antibodies should be stored at -20°C and should NOT BE ALIQUOTED to maintain stability

  • Solution: Follow manufacturer recommendations for storage and handling

• Background reduction:

  • For immunofluorescence, high background can obscure centrosomal/ciliary signals

  • Solution: Optimize blocking conditions (e.g., TBS with 5% milk and 0.1% Tween-20 for Western blots) , and include appropriate controls to distinguish specific from non-specific signals

How can researchers validate SDCCAG8 antibody specificity?

Validating SDCCAG8 antibody specificity is crucial for reliable results:

• Genetic controls:

  • Use SDCCAG8 knockdown or knockout cells as negative controls

  • Demonstrated approach: Antibodies against CEP131 failed to co-immunoprecipitate FLAG-SDCCAG8 from siCEP131 knockdown cell lysates, confirming specificity of the interaction

• Expression controls:

  • Overexpression of tagged SDCCAG8 constructs can provide positive controls

  • Western blot analysis should show bands of expected molecular weight (approximately 83 kDa for full-length SDCCAG8)

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of SDCCAG8

  • Compare results between polyclonal and monoclonal antibodies

  • Example approach: Multiple SDCCAG8 antibodies targeting different regions have been used to confirm the specificity of centrosomal localization

What are emerging research areas involving SDCCAG8?

Based on current findings, several promising research directions for SDCCAG8 include:

• Role in ciliopathies:

  • Further characterization of SDCCAG8 function in ciliary transition zone

  • Investigation of tissue-specific effects of SDCCAG8 mutations

  • Sdccag8^gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog signaling that warrants further investigation

• Protein interaction network:

  • Expanded analysis of the SDCCAG8 interactome across different cell types and conditions

  • Current evidence groups SDCCAG8 interacting proteins into four functional categories: centriolar satellite components, endosomal vesicle components, tRNA synthesis complex proteins, and myosin type II motors involved in ciliogenesis

• Therapeutic targeting:

  • Exploration of strategies to modulate SDCCAG8 function in disease contexts

  • Investigation of small molecules or biologics that could affect SDCCAG8 interactions or function

• Role in distal appendage formation:

  • Despite the loss of cilia in SDCCAG8 knockdown cells, no abnormalities were observed in the formation of centriolar distal appendages (marked by CEP83 and FBF1)

  • This suggests SDCCAG8 functions downstream of distal appendage formation in the ciliogenesis pathway

What methodological advances would benefit SDCCAG8 research?

Several technological and methodological advances could significantly enhance SDCCAG8 research:

• Advanced imaging techniques:

  • Super-resolution microscopy for precise localization studies

  • Live-cell imaging to track SDCCAG8 dynamics during cell cycle and ciliogenesis

• Protein structure analysis:

  • Structural determination of SDCCAG8 and its complexes to understand interaction mechanisms

  • Structure-based drug design targeting SDCCAG8 interactions

• Gene editing approaches:

  • CRISPR/Cas9-mediated generation of isoform-specific knockouts

  • Introduction of patient-specific mutations to model disease states

• Tissue-specific models:

  • Organ-on-chip or organoid systems to study SDCCAG8 function in kidney, retina, and other affected tissues

  • Conditional knockout models to dissect tissue-specific roles

These technological advances would provide deeper insights into SDCCAG8 function and potentially identify new therapeutic targets for SDCCAG8-associated ciliopathies.