SDCCAG3 Antibody

What cellular localization patterns are expected when using SDCCAG3 antibodies?

SDCCAG3 demonstrates distinct localization patterns that researchers should anticipate when designing immunofluorescence experiments. The protein primarily localizes to early and recycling endosomes as well as the basal body of primary cilia . When performing immunofluorescence studies, expect to observe:

• Basal body localization in approximately 10% of ciliated retinal pigment epithelial (RPE) cells

• Higher basal body detection rates in IMCD3 cells (29%) and HEK cells (22%)

• Partial co-localization with centrosomal markers like pericentrin (PCNT)

• Co-localization with EGFP-Arl13b at the ciliary base

For optimal detection, commercial antibodies targeting different epitopes are available, including those recognizing amino acids 263-411 (Sigma) and 188-412 (Proteintech) .

How should SDCCAG3 antibodies be validated for specificity in experimental settings?

Thorough antibody validation is critical for reliable SDCCAG3 detection. Researchers should implement the following validation methods:

• Perform siRNA-mediated knockdown experiments to confirm antibody specificity, as demonstrated in previous studies using multiple independent siRNAs targeting SDCCAG3

• Include appropriate negative controls in immunofluorescence by comparing staining patterns in SDCCAG3-depleted cells versus control cells

• Validate western blot specificity by observing multiple bands (48-60 kDa) due to phosphorylation-dependent mobility shifts

• Cross-validate results using multiple antibodies targeting different epitopes of SDCCAG3 when possible

• For overexpression studies, use EGFP-tagged SDCCAG3 constructs to confirm antibody recognition patterns

What are the recommended protocols for SDCCAG3 antibody applications?

Based on published research methodologies and commercial antibody specifications, the following protocols are recommended:

Western Blotting Protocol:

• Dilution range: 1:500-1:2000

• Expected molecular weight: 48-60 kDa (multiple bands due to phosphorylation)

• Blocking: 5% non-fat milk in TBST buffer

• Primary antibody incubation: Overnight at 4°C

• Detection: Enhanced chemiluminescence after HRP-conjugated secondary antibody incubation

Immunofluorescence Protocol:

• Dilution range: 1:20-1:200

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Co-staining markers: Acetylated tubulin (axoneme), gamma-tubulin (basal body), or pericentrin (centrosome)

• Mounting: Anti-fade medium containing DAPI for nuclear counterstaining

How can SDCCAG3 antibodies be effectively used to study cilia-related functions?

SDCCAG3 plays crucial roles in ciliogenesis, requiring carefully designed experimental approaches:

Recommended Experimental Design:

• Establish baseline cilia formation in your cell model using acetylated tubulin staining

• Design siRNA-mediated knockdown experiments using validated SDCCAG3 siRNAs

• Quantify changes in:

  • Percentage of ciliated cells

  • Ciliary length

  • Localization of cargo proteins (especially Polycystin-2)

• Perform rescue experiments using expression constructs for:

  • Full-length SDCCAG3

  • N-terminal truncation mutant (Δ1-100)

  • N-terminal fragment (aa 1-100)

Previous research demonstrated that SDCCAG3 depletion significantly reduced both the percentage of ciliated cells and ciliary length in multiple cell types. These defects could be rescued by expression of full-length SDCCAG3 but not the N-terminal truncation mutant (Δ1-100) .

What interaction partners should be considered when investigating SDCCAG3 function using antibody-based methods?

SDCCAG3 interacts with several proteins involved in ciliogenesis and membrane trafficking. When designing co-immunoprecipitation or co-localization experiments, consider these key interaction partners:

Primary Interaction Partners:

• IFT88: Intraflagellar transport protein that directly interacts with SDCCAG3 N-terminus (aa 1-100)

  • Interaction region: amino acids 400-550 of IFT88 (containing TPR repeats)

  • Confirmed by: GST-pulldown, yeast two-hybrid, and GFP-pulldown assays

• Polycystin-2: Transmembrane protein whose ciliary localization depends on SDCCAG3

  • Detection method: Anti-polycystin-2 antibody (Santa Cruz, sc-28331, 1:300 dilution)

  • Phenotype: Reduced ciliary localization in SDCCAG3-depleted cells

• Dysbindin: Links SDCCAG3 with ESCRT machinery for receptor sorting

  • Function: Involved in sorting of Fas receptors for lysosomal degradation

• PTPN13: Protein tyrosine phosphatase that forms a complex with SDCCAG3

  • Function: Negative regulator of Fas receptors at early/sorting endosomes

• DLG1: Functions upstream of SDCCAG3 in ciliary protein targeting

  • Phenotype: Reduced ciliary SDCCAG3 in DLG1-depleted cells

How can potential cross-reactivity issues with SDCCAG3 antibodies be addressed in complex tissue samples?

Cross-reactivity is a significant concern when studying SDCCAG3 in complex tissue samples. Consider these approaches to minimize false positive results:

Cross-Reactivity Mitigation Strategies:

• Genetic controls: Generate CRISPR/Cas9 knockout cell lines or tissue-specific conditional knockout models for definitive negative controls

• Absorption controls: Pre-incubate antibody with recombinant SDCCAG3 protein to determine specific versus non-specific binding

• Multiple antibody validation: Use at least two antibodies targeting different epitopes:

  • Anti-SDCCAG3 (Ab1): Proteintech, targeting aa188-412

  • Anti-SDCCAG3 (Ab2): Sigma, targeting aa263-411

• Blocking peptide competition: Perform parallel staining with antibody pre-incubated with immunizing peptide

• Species-specific validation: When working across species, verify antibody reactivity in each species (human, mouse, rat)

What approaches can resolve discrepancies in SDCCAG3 detection between different cellular compartments?

Researchers may encounter variable SDCCAG3 detection depending on cellular localization. To resolve these discrepancies:

Methodological Solutions:

• Subcellular fractionation: Isolate distinct cellular compartments (endosomes, centrosomes, ciliary fraction) prior to western blot analysis

• Proximity ligation assay (PLA): Detect SDCCAG3 interactions with compartment-specific markers with higher sensitivity than conventional immunofluorescence

• Live-cell imaging: Use fluorescently tagged SDCCAG3 constructs to track dynamic localization changes

• Super-resolution microscopy: Employ techniques like STED or STORM to resolve closely positioned compartments such as the transition zone and basal body

• Electron microscopy immunogold labeling: Achieve nanometer-scale resolution of SDCCAG3 localization using specific antibodies

Research has confirmed that SDCCAG3 localization is compartment-specific, with detection rates varying significantly between endosomes (predominant) and ciliary basal bodies (10-29% of ciliated cells depending on cell type) .

How can SDCCAG3 antibodies be effectively used to study protein trafficking pathways?

SDCCAG3 functions in both endosomal sorting and ciliary protein trafficking, requiring specialized methodologies:

Recommended Trafficking Study Approaches:

• Pulse-chase experiments: Use surface biotinylation followed by SDCCAG3 immunoprecipitation to track receptor internalization

• Fluorescent cargo tracking: Monitor trafficking of labeled receptors (e.g., Fas receptor) in the presence or absence of SDCCAG3

• Endosomal marker co-localization: Quantify SDCCAG3 co-localization with:

  • EEA1 (early endosomes)

  • Rab11 (recycling endosomes)

  • LAMP1 (late endosomes/lysosomes)

• IFT cargo analysis: Examine ciliary cargo localization using specific antibodies:

  • Anti-polycystin-2 (Santa Cruz, sc-28331, 1:300 dilution)

  • Anti-Rab8 (Proteintech, 55296-1-AP, 1:200 dilution)

Research has established that SDCCAG3 depletion specifically impairs polycystin-2 trafficking to cilia while leaving Rab8 localization unaffected, suggesting cargo selectivity in SDCCAG3-mediated trafficking .

What analytical parameters should be considered when quantifying SDCCAG3 immunofluorescence in ciliary studies?

Quantitative analysis of SDCCAG3 immunofluorescence requires standardized parameters:

Critical Quantification Parameters:

• Ciliation rate: Calculate percentage of ciliated cells (acetylated tubulin-positive) in multiple fields (minimum 100 cells per condition)

• Ciliary length measurement: Measure axoneme length using specialized software (ImageJ with ciliary length plugin)

• Signal intensity normalization: Normalize SDCCAG3 fluorescence intensity to:

  • Background signal

  • Reference marker (e.g., pericentrin at basal body)

• Co-localization coefficient: Calculate Pearson's or Mander's coefficient for SDCCAG3 with markers like:

  • Pericentrin (basal body)

  • Acetylated tubulin (axoneme)

  • IFT88 (ciliary transition zone)

• Z-stack acquisition: Collect complete z-series (0.3-0.5μm steps) through entire cilium height

Published research demonstrated quantifiable defects upon SDCCAG3 depletion, with ciliary length reduced by approximately 40% and the percentage of ciliated cells decreased by 50-60% in SDCCAG3 knockdown cells compared to controls .

What are the most common technical challenges when using SDCCAG3 antibodies for immunoprecipitation studies?

Researchers frequently encounter technical difficulties when performing SDCCAG3 immunoprecipitation. Address these challenges with the following strategies:

Immunoprecipitation Optimization Strategies:

• Lysis buffer optimization: Use buffers containing:

  • 1% NP-40 or Triton X-100

  • 150mM NaCl

  • 50mM Tris-HCl (pH 7.4)

  • Protease and phosphatase inhibitors

• Cross-linking consideration: Implement DSP or formaldehyde cross-linking to capture transient interactions

• Pre-clearing step: Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Antibody selection: Use antibodies validated for immunoprecipitation applications

• Co-factor addition: Include ATP (1-5mM) to stabilize certain protein interactions

• Detergent sensitivity: Test multiple detergent conditions as some interactions may be detergent-sensitive

For confirming IFT88-SDCCAG3 interactions, previous research successfully employed reciprocal co-immunoprecipitation approaches using both Myc-SDCCAG3 and EGFP-IFT88 constructs .

How can phosphorylation status affect SDCCAG3 antibody detection, and how should researchers account for this?

SDCCAG3 undergoes multiple phosphorylation events that can influence antibody detection and protein mobility:

Phosphorylation-Related Considerations:

• Western blot pattern: Expect multiple bands (48-60 kDa) representing different phosphorylation states

• Phosphatase treatment: Include controls with lambda phosphatase treatment to confirm phosphorylation-dependent mobility shifts

• Phospho-specific antibodies: Consider developing phospho-specific antibodies for key regulatory sites

• Phosphomimetic mutants: Use phosphomimetic (S/T→D/E) or phospho-dead (S/T→A) mutants to study functional relevance

• Cell cycle synchronization: Synchronize cells when studying cell-cycle dependent phosphorylation events

Previous studies have documented that SDCCAG3 typically appears as multiple bands in western blots due to phosphorylation, which can complicate interpretation of results if not properly controlled .

What considerations should guide the selection of cell models for SDCCAG3 antibody-based research?

Cell model selection significantly impacts SDCCAG3 antibody-based research outcomes:

Cell Model Selection Guidelines:

• Ciliated cell lines: Prioritize naturally ciliated cell types:

  • RPE cells (retinal pigment epithelial)

  • IMCD3 cells (inner medullary collecting duct)

  • HEK293 cells (human embryonic kidney)

• Expression level verification: Confirm endogenous SDCCAG3 expression levels by western blot

• Ciliary induction conditions: Standardize serum starvation protocols (typically 24-48h) to induce primary cilia

• Species considerations: Match antibody reactivity (human, mouse, rat) to your model

• Tissue relevance: Select models relevant to SDCCAG3 function (kidney cells for polycystic kidney disease studies)

Research has demonstrated variable basal body localization rates of SDCCAG3 across different cell types: 10% in RPE cells, 29% in IMCD3 cells, and 22% in HEK cells , highlighting the importance of cell model selection.

What emerging methodologies might enhance SDCCAG3 antibody-based research?

Emerging technologies offer new opportunities for advancing SDCCAG3 research:

Innovative Methodological Approaches:

• Proximity labeling: Employ BioID or TurboID-SDCCAG3 fusion proteins to identify spatial interaction networks at endosomes versus cilia

• Optogenetic manipulation: Develop light-inducible SDCCAG3 degradation systems to study acute loss-of-function

• Live super-resolution imaging: Track SDCCAG3 dynamics during ciliogenesis using lattice light-sheet microscopy

• CRISPR-based tagging: Generate endogenously tagged SDCCAG3 to avoid overexpression artifacts

• Single-molecule tracking: Analyze SDCCAG3 movement between cellular compartments using quantum dot-labeled antibodies

• Intrabodies: Develop cell-permeable antibody fragments for live-cell visualization without fixation

Recent advanced approaches include proximity-dependent biotinylation techniques that have identified SDCCAG3 in the interactome of several centriolar proteins .

How might antibody-based studies contribute to understanding SDCCAG3's dual role in ciliogenesis and apoptosis regulation?

SDCCAG3's multiple cellular functions present unique research opportunities:

Integrated Research Approaches:

• Conditional domain deletion: Generate domain-specific knockouts to separate cilia versus apoptotic functions

• Pathway-specific interactome analysis: Perform immunoprecipitation followed by mass spectrometry under conditions that favor:

  • Ciliogenesis (serum starvation)

  • Apoptosis (Fas ligand stimulation)

• Domain-specific antibodies: Develop antibodies targeting functional domains:

  • N-terminal domain (aa 1-100): IFT88 binding, ciliary function

  • Central/C-terminal regions: Endosomal sorting, apoptosis regulation

• Phosphorylation-state specific analysis: Examine how phosphorylation regulates SDCCAG3's distribution between ciliary versus endosomal functions

Studies have established that SDCCAG3 regulates both Fas receptor trafficking in apoptotic pathways and polycystin-2 trafficking in ciliary pathways, suggesting compartmentalized functions that could be investigated through domain-specific approaches .

What strategies might enhance antibody-based detection of low-abundance SDCCAG3 in primary tissues and patient samples?

Clinical and primary tissue studies require enhanced detection methods:

Enhanced Detection Strategies:

• Signal amplification: Implement tyramide signal amplification (TSA) to enhance immunohistochemical detection

• Multiplex immunofluorescence: Combine SDCCAG3 detection with tissue-specific markers using spectral unmixing

• RNAscope-immunofluorescence combination: Correlate mRNA expression with protein localization

• Ultrasensitive ELISA development: Design sandwich ELISA with optimized antibody pairs for detection in tissue lysates

• Tissue clearing techniques: Apply CLARITY or iDISCO for whole-mount 3D visualization of SDCCAG3 distribution

• Single-cell western blot: Detect SDCCAG3 in rare cell populations using microfluidic single-cell western blot

These advanced detection methods could facilitate translation of SDCCAG3 research findings to clinical samples, particularly in polycystic kidney disease where SDCCAG3's role in polycystin-2 trafficking has direct disease relevance .

What quantitative data exists regarding SDCCAG3 depletion effects on ciliogenesis?

The following table summarizes key quantitative findings from published research on SDCCAG3's role in ciliogenesis:

These quantitative measurements demonstrate that SDCCAG3 depletion significantly impacts both cilia formation and length, effects that can be rescued by full-length SDCCAG3 but not by the N-terminal truncation mutant .

What structural and interaction mapping data exists for SDCCAG3 protein domains?

Research has defined key structural domains of SDCCAG3 and their interactions:

The N-terminal domain (aa 1-100) has been identified as both necessary and sufficient for basal body localization and IFT88 interaction, while the C-terminal regions appear more involved in endosomal functions .

What antibody-specific validation data supports SDCCAG3 detection specificity?

Multiple validation approaches confirm the specificity of SDCCAG3 antibodies:

These validation approaches collectively demonstrate antibody specificity and provide guidelines for appropriate experimental conditions when using SDCCAG3 antibodies .